A role for the ventral hippocampal endocannabinoid system in fear-conditioned analgesia and fear responding in the presence of nociceptive tone in rats.

The endogenous cannabinoid (endocannabinoid) system plays an important role in fear-conditioned analgesia (FCA) and expression and extinction of conditioned fear. The hippocampus has an established role in both pain and conditioned fear and is a substrate for endocannabinoid activity. This study aimed to investigate the role of the endocannabinoid system in the ventral hippocampus (vHip) in FCA and in fear responding in the presence of nociceptive tone. Fear-conditioned rats displayed significantly increased freezing and 22-kHz ultrasonic vocalisation and a reduction in formalin-evoked nociceptive behaviour (ie, FCA) upon re-exposure to a context previously paired with footshock. Tissue levels of the endocannabinoids, anandamide, and 2-arachidonoylglycerol, as well as the fatty acid amide, palmitoylethanolamide, were significantly higher in the vHip of fear-conditioned rats compared with non-fear-conditioned controls. URB597 (inhibitor of fatty acid amide hydrolase [FAAH]), administered bilaterally into the vHip, significantly enhanced FCA during the entire trial and increased fear responding in formalin-treated rats early in the trial. The URB597-induced enhancement of FCA was blocked by intra-vHip administration of the cannabinoid(1) (CB(1)) receptor antagonist/inverse agonist rimonabant. Intra-vHip rimonabant alone had no effect on the expression of FCA, and URB597 did not significantly alter formalin-evoked nociceptive behaviour in non-fear-conditioned rats. These data suggest an important role for the endocannabinoid system in the vHip in FCA, whereby levels of 2-arachidonoylglycerol and the FAAH substrates palmitoylethanolamide and anandamide are increased in rats expressing FCA, and pharmacological inhibition of FAAH in the vHip enhances this form of endogenous analgesia via a CB(1) receptor-dependent mechanism.peer-reviewe

The endocannabinoid system in the rat dorsolateral periaqueductal grey mediates fear-conditioned analgesia and controls fear expression in the presence of nocicpetive tone

Background and purpose: Endocannabinoids in the midbrain periaqueductal grey (PAG) are involved in modulating nociception and unconditioned stress-induced analgesia, however, their role in fear-conditioned analgesia (FCA) has not been examined. The present study examined the role of the endocannabinoid system in the dorsolateral (dl) PAG in formalin-evoked nociceptive behaviour, conditioned fear and FCA in rats. Experimental approach: Rats received intra-dlPAG administration of the CB1 receptor antagonist/inverse agonist rimonabant, or vehicle, prior to re-exposure to a context paired 24hrs previously with footshock. Formalin-evoked nociceptive behaviour and fear-related behaviours (freezing and 22kHz ultrasonic vocalisation) were assessed. In a separate cohort, alterations in levels of endocannabinoids (2-arachidonoyl glycerol [2-AG] and N-arachidonoyl ethanolamide [anandamide; AEA]) and the related N-acylethanolamines (NAEs) (N-palmitoyl ethanolamide [PEA] and N-oleoyl ethanolamide [OEA]) were measured in dlPAG tissue following re-exposure to conditioned context in the presence or absence of formalin-evoked nociceptive tone. Key results: Re-exposure of rats to the context previously associated with footshock resulted in FCA. Intra-dlPAG administration of rimonabant significantly attenuated FCA and fear-related behaviours expressed in the presence of nociceptive tone. Conditioned fear in the absence of formalin-evoked nociceptive tone was associated with increased levels of the endocannabinoids (2-AG and AEA) and NAEs (PEA and OEA) in the dlPAG. FCA was specifically associated with an increase in AEA levels in the dlPAG. Conclusions and implications: These data suggest that conditioned fear to context mobilises endocannabinoids and NAEs in the dlPAG and support a role for the endocannabinoid system in the dlPAG in mediating the potent suppression of pain responding which occurs during exposure to conditioned aversive contexts.peer-reviewe

Involvement of the endocannabinoid system in attentional modulation of nociceptive behaviour in rats

BackgroundDistraction is used clinically to relieve and manage pain. It is hypothesized that pain demands attention and that exposure to another attention-demanding stimulus causes withdrawal of attention away from painful stimuli, thereby reducing perceived pain. We have recently developed a rat model that provides an opportunity to investigate the neurobiological mechanisms mediating distraction-induced analgesia, as these mechanisms are, at present, poorly understood. Given the well-described role of the endogenous cannabinoid (endocannabinoid; EC) system in the modulation of pain and attentional processing, the present study investigated its role in distraction-induced antinociception in rats.MethodsAnimals received the CB1 receptor antagonist/inverse agonist, rimonabant or vehicle intraperitoneally, 30min prior to behavioural evaluation. Formalin-evoked nociceptive behaviour was measured in the presence or absence of a novel-object distractor. Liquid chromatography-tandem mass spectrometry was used to determine the levels of the endogenous cannabinoids anandamide and 2-arachidonoylglycerol (2-AG) in the ventral hippocampus (vHip).ResultsExposure to a novel object distractor significantly reduced formalin-evoked nociceptive behaviour. The novel object-induced reduction in nociceptive behaviour was attenuated by rimonabant. Novel object exposure was also associated with increased tissue levels of anandamide and 2-AG in the vHip.ConclusionsThese data suggest that the reduction in formalin-evoked nociceptive behaviour that occurs as a result of exposure to a novel object may be mediated by engagement of the EC system, in particular in the vHip. The results provide evidence that the EC system may be an important neural substrate subserving attentional modulation of pain.This publication has emanated from research conducted with the financial support of the Science Foundation Ireland under Grant Numbers 05/Y12/B686 and 10/IN.1/B2976.peer-reviewe

Pharmacological inhibition of monoacylglycerol lipase attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action

Background and purpose: This study determined the effect of JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme which preferentially catabolises the endocannabinoid 2-arachidonoyl glycerol (2-AG), on inflammatory cytokines in the brain and plasma following an acute immune challenge. The receptor and molecular mechanisms involved were also investigated.Experimental approach: JZL184 and/or AM251 (CB1 antagonist) or AM630 (CB2 antagonist) were administered to rats 30 min prior to the administration of lipopolysaccharide (LPS), 2hrs following which cytokine expression/levels, MAGL activity, 2-AG, arachidonic acid and prostaglandin levels were measured in the frontal cortex, plasma and spleen.Key Results: JZL184 attenuated LPS-induced increases in IL-1β, IL-6, TNF-α and IL-10, but not IĸBα expression in the rat frontal cortex. AM251 attenuated the JZL184-induced decrease in frontal cortical IL-1β expression. Although arachidonic acid levels in the frontal cortex were reduced in JZL184-treated rats, MAGL activity, 2-AG, PGE2 and PGD2 levels remained unchanged. In comparison, MAGL activity was inhibited and 2-AG levels enhanced in the spleen following JZL184 administration. In the plasma, LPS-induced increases in TNF-α and IL-10 levels were attenuated by JZL184, an effect partially blocked by AM251. In addition, AM630 blocked the LPS-induced increases in plasma IL-1β in the presence, but not absence, of JZL184.Conclusion and Implications: Inhibition of peripheral MAGL in the rat by JZL184 suppresses LPS-induced circulating cytokines which in turn may modulate central cytokine expression. The data provide further evidence for the therapeutic potential of targeting the endocannabinoid system for the treatment of central and peripheral inflammatory disorders.peer-reviewe

Cdc7-dependent and -independent phosphorylation of claspin in the induction of the dna replication checkpoint

Claspin is a critical mediator protein in the DNA replication checkpoint, responsible for ATR-dependent activation of the effector kinase Chk1. Cdc7, an essential kinase required for the initiation of DNA replication, can also interact with and phosphorylate Claspin. In this study we use small-molecule inhibitors of Cdc7 kinase to further understand the relationship between Cdc7, Claspin and Chk1 activation. We demonstrate that inhibition of Cdc7 kinase delays HU-induced phosphorylation of Chk1 but does not affect the maintenance of the replication checkpoint once it is established. We find that while chromatin association of Claspin is not affected by Cdc7 inhibition, Claspin phosphorylation is attenuated following HU treatment, which may be responsible for the altered kinetics of HU-induced Chk1 phosphorylation. We demonstrate that Claspin is an in vitro substrate of Cdc7 kinase, and using mass-spectrometry, we identify multiple phosphorylation sites that help to define a Cdc7 phosphorylation motif. Finally, we show that the interaction between Claspin and Cdc7 is not dependent on Cdc7 kinase activity, but Claspin interaction with the DNA helicase subunit Mcm2 is lost upon Cdc7 inhibition. We propose Cdc7-dependent phosphorylation regulates critical protein-protein interactions and modulates Claspin's function in the DNA replication checkpoint

Cdc7-dependent and -independent phosphorylation of claspin in the induction of the dna replication checkpoint

Claspin is a critical mediator protein in the DNA replication checkpoint, responsible for ATR-dependent activation of the effector kinase Chk1. Cdc7, an essential kinase required for the initiation of DNA replication, can also interact with and phosphorylate Claspin. In this study we use small-molecule inhibitors of Cdc7 kinase to further understand the relationship between Cdc7, Claspin and Chk1 activation. We demonstrate that inhibition of Cdc7 kinase delays HU-induced phosphorylation of Chk1 but does not affect the maintenance of the replication checkpoint once it is established. We find that while chromatin association of Claspin is not affected by Cdc7 inhibition, Claspin phosphorylation is attenuated following HU treatment, which may be responsible for the altered kinetics of HU-induced Chk1 phosphorylation. We demonstrate that Claspin is an in vitro substrate of Cdc7 kinase, and using mass-spectrometry, we identify multiple phosphorylation sites that help to define a Cdc7 phosphorylation motif. Finally, we show that the interaction between Claspin and Cdc7 is not dependent on Cdc7 kinase activity, but Claspin interaction with the DNA helicase subunit Mcm2 is lost upon Cdc7 inhibition. We propose Cdc7-dependent phosphorylation regulates critical protein-protein interactions and modulates Claspin\u27s function in the DNA replication checkpoint

Negative ion cid fragmentation of o-linked oligosaccharide aldoses—charge induced and charge remote fragmentation

Collision induced dissociation (CID) fragmentation was compared between reducing and reduced sulfated, sialylated, and neutral O-linked oligosaccharides. It was found that fragmentation of the [M - H](-) ions of aldoses with acidic residues gave unique Z-fragmentation of the reducing end GalNAc containing the acidic C-6 branch, where the entire C-3 branch was lost. This fragmentation pathway, which is not seen in the alditols, showed that the process involved charge remote fragmentation catalyzed by a reducing end acidic anomeric proton. With structures containing sialic acid on both the C-3 and C-6 branch, the [M - H](-) ions were dominated by the loss of sialic acid. This fragmentation pathway was also pronounced in the [M - 2H](2-) ions revealing both the C-6 Z-fragment plus its complementary C-3 C-fragment in addition to glycosidic and cross ring fragmentation. This generation of the Z/C-fragment pairs from GalNAc showed that the charges were not participating in their generation. Fragmentation of neutral aldoses showed pronounced Z-fragmentation believed to be generated by proton migration from the C-6 branch to the negatively charged GalNAc residue followed by charge remote fragmentation similar to the acidic oligosaccharides. In addition, A-type fragments generated by charge induced fragmentation of neutral oligosaccharides were observed when the charge migrated from C-1 of the GalNAc to the GlcNAc residue followed by rearrangement to accommodate the (0,2)A-fragmentation. LC-MS also showed that O-linked aldoses existed as interchangeable alpha/beta pyranose anomers, in addition to a third isomer (25% of the total free aldose) believed to be the furanose form