Anne M. Zimmer

Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB2 receptor

Cannabinoids have immunomodulatory as well as psychoactive effects. Because the central cannabinoid receptor (cannabinoid CB(1) receptor) is highly expressed in many neuronal tissues and the peripheral cannabinoid receptor (cannabinoid CB(2) receptor) is highly expressed in immune cells, it has been suggested that the central nervous system effects of cannabinoids are mediated by cannabinoid CB(1) receptors and that the immune effects are mediated by cannabinoid CB(2) receptors. To test this hypothesis, we have generated the first mouse strain with a targeted mutation in the cannabinoid CB(2) receptor gene. Binding studies using the highly specific synthetic cannabinoid receptor agonist (-)-cis-3-¿2-Hydroxy-4-(1, 1-dimethylheptyl)phenyl-trans-4-(3-hydroxypropyl)cyclohexanol (¿3HCP 55,940) revealed no residual cannabinoid binding sites in the spleen of the cannabinoid CB(2) receptor knockout mice, while binding in the central nervous system was unchanged. Cannabinoid CB(2) receptor knockout mice, which appear healthy, are fertile and care for their offspring. Fluorescence activated cell sorting (FACS) analysis showed no differences in immune cell populations between cannabinoid CB(2) receptor knockout and wildtype mice. We investigated the immunomodulatory effects of cannabinoids in cannabinoid CB(2) receptor deficient mice using a T cell co-stimulation assay. Delta(9)Tetrahydrocannabinol inhibits helper T cell activation through macrophages derived from wild type, but not from knockout mice, thus indicating that this effect is mediated by the cannabinoid CB(2) receptor. In contrast, central nervous system effects of cannabinoids were not altered in these mice. Our results suggest that cannabinoid CB(2) receptor-specific ligands may be clinically useful in the modulation of macrophage immune function while exhibiting no central nervous system activity. Furthermore, we conclude that the cannabinoid CB(2) receptor knockout mouse is a useful animal model in which to study the role of the cannabinoid system in immunoregulation.

Levels, metabolism, and pharmacological activity of anandamide in CB1 cannabinoid receptor knockout mice: Evidence for non-CB1, non-CB2 receptor-mediated actions of anandamide in mouse brain

Abstract: 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.

A role for ASIC3 in the modulation of high-intensity pain stimuli

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.

A tarantula peptide against pain via ASIC1a channels and opioid mechanisms

Psalmotoxin 1, a peptide extracted from the South American tarantula Psalmopoeus cambridgei, has very potent analgesic properties against thermal, mechanical, chemical, inflammatory and neuropathic pain in rodents. It exerts its action by blocking acid-sensing ion channel 1a, and this blockade results in an activation of the endogenous enkephalin pathway. The analgesic properties of the peptide are suppressed by antagonists of the μ and δ-opioid receptors and are lost in Penk1−/− mice.

Craf-1 protein kinase is essential for mouse development

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from a variety of cell surface receptors to the nucleus. We report here that Craf-1 is essential for mouse development, as its mutation results in embryonic lethality. Developmental defects are found in mutant placentas as well as in the skin and in the lungs of mutant embryos. Craf-1 mutants also display a generalized growth retardation which is consistent with the ubiquitous expression of Craf-1 and which could be due to the reduced proliferation of mutant cells. Interestingly, the time-point of embryonal death varies depending on the genetic background. This suggests that Craf-1-mediated signaling is affected by genetic background-specific alleles of other genes.

Protective role of palmitoylethanolamide in contact allergic dermatitis

To cite this article: Petrosino S, Cristino L, Karsak M, Gaffal E, Ueda N, Tüting T, Bisogno T, De Filippis D, D’Amico A, Saturnino C, Orlando P, Zimmer A, Iuvone T, Di Marzo V. Protective role of palmitoylethanolamide in contact allergic dermatitis. Allergy 2010; 65: 698–711.

Control of hormonal stress reactivity by the endogenous opioid system.

Regulations of hormonal stress responses entail the initiation, amplitude and termination of the reaction, as well as its integration with other stress response systems. This study investigates the role of endogenous opioids in the regulation and integration of behavioral, thermal and hormonal stress responses, as these neuromodulators and their receptors are expressed in limbic structures responsible for stress responses. For this purpose, we subjected mice with selective deletion of beta-endorphin, enkephalin or dynorphin to the zero-maze test, a mildly stressful situation, and registered behaviors and stress hormone levels. Behavioral stress reactivity was assessed using zero-maze, light-dark and startle-reactivity paradigms. Animals lacking enkephalin displayed increased anxiety-related behavioral responses in each three, dynorphin knockouts in two models, whereas the responses of beta-endorphin knockouts indicated lower anxiety level in the zero-maze test. All knockout strains showed marked changes in hormonal stress reactivity. Increase in ACTH level after zero-maze test situation, unlike in wild type animals, failed to reach the level of significance in Penk1(-/-) and Pdyn(-/-) mice. Corticosterone plasma levels rapidly increased in all strains, with a lower peak response in knockouts. In wild-type and beta-endorphin-deficient mice, corticosterone levels returned to baseline within 60min after stress exposure. In contrast, mice lacking dynorphin and enkephalin showed longer-lasting elevated corticosterone levels, indicating a delayed termination of the stress reaction. Importantly, the behavioral and hormonal responses correlated in wild-type but not in knockout mice. Hyperthermia elicited by stress was reduced in animals lacking dynorphin and absent in Penk1(-/-) mice, despite of the heightened behavioral anxiety level of these strains. These results demonstrate an important role on the endogenous opioid system in the integration of behavioral and hormonal stress responses.

Behavioral phenotype of pre-proenkephalin-deficient mice on diverse congenic backgrounds

RationaleThe phenotype of genetically modified animals is thought to result from an interaction of gene manipulation with the genetic background and environmental factors.ObjectivesTo test the behavioral and drug responses of Penk1−/− mice on different genetic backgrounds.MethodsCongenic C57BL/6J and DBA/2J mouse strains with a targeted deletion of the Penk1 gene were generated. Behavior and drug effects were tested in models of pain and anxiety.ResultsPenk1−/− mice showed exaggerated responses to painful or threatening environmental stimuli, but the expressivity of the mutant phenotype was strongly dependent on the behavioral paradigm and on the genetic background. For example, elevated levels of anxiety were readily detectable in C57BL/6J-Penk1−/− mice in the light–dark and startle response tests, but not in the social interaction test. In contrast, we found elevated levels of anxiety in DBA/2J-Penk1−/− mice only in the zero-maze and social interaction tests. In some cases, the idiosyncratic behavior masked the appearance of the knockout gene effect. The activity of the anxiogenic drug, m-chlorophenylpiperazine, but not the anxiolytic drug diazepam, was strain and genotype dependent. Mice with the Penk1 mutation on the DBA/2J, but not on other genetic backgrounds, showed an increased opioid-dependent stress-induced analgesia.Conclusions(1) The behavioral effects of the Penk1 gene deletion persists on different genetic backgrounds, but its detection sometimes requires the use of different behavioral paradigms. (2) The behavior of the background strain should be considered in the analysis of knockout mice to avoid floor and ceiling effects, which may mask the phenotype.

Region selective up-regulation of μ-, δ- and κ-opioid receptors but not opioid receptor-like 1 receptors in the brains of enkephalin and dynorphin knockout mice

Abstract The role of endogenous opioid peptides and receptors has recently been investigated using knockout mice. Although the affinities of opioid peptides for opioid receptors has been known for many years there is still some uncertainty over which receptor is the endogenous target for each peptide. To address this issue we have studied using quantitative autoradiography the levels of all four opioid receptor subtypes (μ, δ, κ and opioid receptor-like 1 [ORL1]) in brains sectioned from enkephalin and dynorphin knockouts, as well as from double knockouts. Because receptor up-regulation has been observed when its cognate ligand-peptide is genetically ablated, regional changes in receptor binding in knockout mice may reflect areas where the peptide ligand is tonically active at its receptor or played a role in receptor regulation. In addition, the study aimed to correlate previously observed behaviour in these animals with receptor modulation. Marked region-specific up-regulation of the μ, δ, and κ opioid receptors but not ORL1 receptors was observed in proenkephalin and prodynorphin knockouts. In proenkephalin knockouts this was most pronounced for the μ- and δ-receptor and in prodynorphin knockouts for the κ-receptor. Combinatorial double knockouts did not show any changes in addition to those observed in single knockouts. The largest changes were observed in limbic regions and our results suggest that proenkephalin peptides are tonically active at μ and δ-receptors predominantly in these areas. Prodynorphin peptides appear to regulate mostly the κ-receptor but they are also modulators of μ- and δ-receptors.

Preprodynorphin mediates locomotion and D2 dopamine and μ‐opioid receptor changes induced by chronic ‘binge’ cocaine administration

Evidence suggests that the κ‐opioid receptor (KOP‐r) system plays an important role in cocaine addiction. Indeed, cocaine induces endogenous KOP activity, which is a mechanism that opposes alterations in behaviour and brain function resulting from repeated cocaine use. In this study, we have examined the influence of deletion of preprodynorphin (ppDYN) on cocaine‐induced behavioural effects and on hypothalamic‐pituitary‐adrenal axis activity. Furthermore, we have measured μ‐opioid receptor (MOP‐r) agonist‐stimulated [35S]GTPγS, dopamine D1, D2 receptor and dopamine transporter (DAT) binding. Male wild‐type (WT) and ppDYN knockout (KO) mice were injected with saline or cocaine (45 mg/kg/day) in a ‘binge’ administration paradigm for 14 days. Chronic cocaine produced an enhancement of locomotor sensitisation in KO. No genotype effect was found on stereotypy behaviour. Cocaine‐enhanced MOP‐r activation in WT but not in KO. There was an overall decrease in D2 receptor binding in cocaine‐treated KO but not in WT mice. No changes were observed in D1 and DAT binding. Cocaine increased plasma corticosterone levels in WT but not in KO. The data confirms that the endogenous KOP system inhibits dopamine neurotransmission and that ppDYN may mediate the enhancement of MOP‐r activity and the activation of the hypothalamic‐pituitary‐adrenal axis after chronic cocaine treatment.