Brendan Harhen

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

BACKGROUND AND PURPOSE Endocannabinoids in the midbrain periaqueductal grey (PAG) modulate 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.

Pharmacological inhibition of endocannabinoid degradation modulates the expression of inflammatory mediators in the hypothalamus following an immunological stressor

The endocannabinoid system is an important regulator of the nervous, neuroendocrine, and immune systems, thus representing a novel therapeutic target for stress-related neuroinflammatory and psychiatric disorders. However, there is a paucity of data relating to the effects of endocannabinoids on neuroinflammatory mediators following an immune stress/challenge in vivo. This study investigated the effects of URB597, a selective inhibitor of fatty acid amide hydrolyase (FAAH), the enzyme that preferentially metabolizes anandamide, on lipopolysaccharide (LPS)-induced increases in the expression of immune mediators in the hypothalamus. Systemic administration of URB597 increased the levels of anandamide and the related N-acylethanolamines, N-palmitoylethanolamide, and N-oleoylethanolamide, but not 2-arachidonoyl glycerol, in the hypothalamus and spleen. URB597 attenuated the LPS-induced increase in interleukin (IL)-1β expression while concurrently augmenting the LPS-induced increase in suppressor of cytokine signalling (SOCS)-3 expression. In addition, URB597 tended to enhance and reduce the LPS-induced increase in IL-6 and IL-10 mRNA expression, respectively. LPS-induced increases in peripheral cytokine levels or plasma corticosterone were not altered by URB597. The present study provides evidence for a role for FAAH in the regulation of LPS-induced expression of inflammatory mediators in the hypothalamus. Improved understanding of endocannabinoid-mediated regulation of neuroimmune function has fundamental physiological and potential therapeutic significance in the context of stress-related disorders.

The monoacylglycerol lipase inhibitor JZL184 attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action.

JZL184 is a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme that preferentially catabolizes the endocannabinoid 2‐arachidonoyl glycerol (2‐AG). Here, we have studied the effects of JZL184 on inflammatory cytokines in the brain and plasma following an acute immune challenge and the underlying receptor and molecular mechanisms involved.

DNA mediated chromatin pull-down for the study of chromatin replication

Chromatin replication involves duplicating DNA while maintaining epigenetic information. These processes are critical for genome stability and for preserving cell-type identity. Here we describe a simple experimental approach that allows chromatin to be captured and its content analysed after in vivo replication and labeling of DNA by cellular DNA polymerases. We show that this technique is highly specific and that proteins bound to the replicated DNA can be analyzed by both immunological techniques and large scale mass spectrometry. As proof of concept we have used this novel procedure to begin investigating the relationship between chromatin protein composition and the temporal programme of DNA replication in human cells. It is expected that this technique will become a widely used tool to address how chromatin proteins assemble onto newly replicated DNA after passage of a replication fork and how chromatin maturation is coupled to DNA synthesis.

Impaired endocannabinoid signalling in the rostral ventromedial medulla underpins genotype-dependent hyper-responsivity to noxious stimuli.

Summary Impaired endocannabinoid signalling in the rostral ventromedial medulla underpins hyper‐responsivity to a noxious inflammatory stimulus in the Wistar–Kyoto rat, a genetic background prone to heightened stress/affect. ABSTRACT Pain is both a sensory and an emotional experience, and is subject to modulation by a number of factors including genetic background modulating stress/affect. The Wistar–Kyoto (WKY) rat exhibits a stress‐hyper‐responsive and depressive‐like phenotype and increased sensitivity to noxious stimuli, compared with other rat strains. Here, we show that this genotype‐dependent hyperalgesia is associated with impaired pain‐related mobilisation of endocannabinoids and transcription of their synthesising enzymes in the rostral ventromedial medulla (RVM). Pharmacological blockade of the Cannabinoid1 (CB1) receptor potentiates the hyperalgesia in WKY rats, whereas inhibition of the endocannabinoid catabolising enzyme, fatty acid amide hydrolase, attenuates the hyperalgesia. The latter effect is mediated by CB1 receptors in the RVM. Together, these behavioural, neurochemical, and molecular data indicate that impaired endocannabinoid signalling in the RVM underpins hyper‐responsivity to noxious stimuli in a genetic background prone to heightened stress/affect.

Evidence for a role of GABAergic and glutamatergic signalling in the basolateral amygdala in endocannabinoid-mediated fear-conditioned analgesia in rats

Summary CB1 receptors in the basolateral amygdala facilitate the expression of fear‐conditioned analgesia through a mechanism involving the modulation of GABAergic and glutamatergic signalling. Abstract The basolateral amygdala (BLA) is a key substrate facilitating the expression of fear‐conditioned analgesia (FCA). However, the neurochemical mechanisms in the BLA which mediate this potent suppression of pain responding during fear remain unknown. The present study investigated the role of cannabinoid1 (CB1) receptors and interactions with GABAergic (GABAA receptor) and glutamatergic (metabotropic glutamate receptor type 5; mGluR5) signalling in the BLA in formalin‐evoked nociceptive behaviour and FCA in rats. Reexposure to a context previously paired with foot shock significantly reduced formalin‐evoked nociceptive behaviour. Systemic or intra‐BLA microinjection of the CB1 receptor antagonist/inverse agonist AM251 prevented this expression of FCA, while injection of AM251 into the central nucleus of the amygdala did not. The suppression of FCA by systemic AM251 administration was partially attenuated by intra‐BLA administration of either the GABAA receptor antagonist bicuculline or the mGluR5 antagonist 2‐methyl‐6‐(phenylethynyl) pyridine, (MPEP). Bilateral microinjection of MPEP, but not bicuculline, alone into the BLA enhanced formalin‐evoked nociceptive behaviour. Postmortem analyses revealed that FCA was associated with a significant increase in tissue levels of anandamide in the BLA side contralateral to intraplantar formalin injection. In addition, fear‐conditioned rats exhibited a robust formalin‐induced increase in levels of 2‐arachidonyl glycerol and N‐palmitoylethanolamide in the ipsilateral and contralateral BLA, respectively. These data suggest that CB1 receptors in the BLA facilitate the expression of FCA, through a mechanism which is likely to involve the modulation of GABAergic and glutamatergic signalling.

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

Summary Endocannabinoid signalling in the rat ventral hippocampus mediates pain suppression during conditioned fear and modulates expression of fear in the presence of nociceptive tone. ABSTRACT 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 cannabinoid1 (CB1) 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 CB1 receptor‐dependent mechanism.

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.

N-palmitoylethanolamide in the anterior cingulate cortex attenuates inflammatory pain behaviour indirectly via a CB1 receptor-mediated mechanism.

Abstract The neural substrates and mechanisms mediating the antinociceptive effects of the endogenous bioactive lipid, N-palmitoylethanolamide (PEA), require further investigation. We investigated the effects of exogenous PEA administration into the anterior cingulate cortex (ACC), an important brain region linked with cognitive and affective modulation of pain, on formalin-evoked nociceptive behaviour in rats. Potential involvement of peroxisome proliferator-activated receptor isoforms (PPAR) &agr; and &ggr; or endocannabinoid-mediated entourage effects at cannabinoid1 (CB1) receptors or transient receptor potential subfamily V member 1 (TRPV1) in mediating the effects of PEA was also investigated. Intra-ACC administration of PEA significantly attenuated the first and early second phases of formalin-evoked nociceptive behaviour. This effect was attenuated by the CB1 receptor antagonist AM251, but not by the PPAR&agr; antagonist GW6471, the PPAR&ggr; antagonist GW9662, or the TRPV1 antagonist 5′-iodo resiniferatoxin. All antagonists, administered alone, significantly reduced formalin-evoked nociceptive behaviour, suggesting facilitatory/permissive roles for these receptors in the ACC in inflammatory pain. Post-mortem tissue analysis revealed a strong trend for increased levels of the endocannabinoid anandamide in the ACC of rats that received intra-ACC PEA. Expression of c-Fos, a marker of neuronal activity, was significantly reduced in the basolateral nucleus of the amygdala, but not in the central nucleus of the amygdala, the rostral ventromedial medulla or the dorsal horn of the spinal cord. In conclusion, these data indicate that PEA in the ACC can reduce inflammatory pain-related behaviour, possibly via AEA-induced activation of CB1 receptors and associated modulation of neuronal activity in the basolateral amygdala.

Microinjection of 2-arachidonoyl glycerol into the rat ventral hippocampus differentially modulates contextually induced fear, depending on a persistent pain state

The endogenous cannabinoid (endocannabinoid) system plays a key role in the modulation of aversive and nociceptive behaviour. The components of the endocannabinoid system are expressed throughout the hippocampus, a brain region implicated in both conditioned fear and pain. In light of evidence that pain can impact on the expression of fear‐related behaviour, and vice versa, we hypothesised that exogenous administration of the endocannabinoid 2‐arachidonoyl glycerol (2‐AG) into the ventral hippocampus (vHip) would differentially regulate fear responding in the absence vs. the presence of formalin‐evoked nociceptive tone. Fear‐conditioned rats showed significantly increased freezing and a reduction in formalin‐evoked nociceptive behaviour upon re‐exposure to a context previously paired with footshock. Bilateral microinjection of 2‐AG into the vHip significantly reduced contextually induced freezing in non‐formalin‐treated rats, and reduced formalin‐evoked nociceptive behaviour in non‐fear‐conditioned rats. In contrast, 2‐AG microinjection had no effect on fear responding in formalin‐treated rats, and no effect on nociceptive behaviour in fear‐conditioned rats. The inhibitory effect of 2‐AG on fear‐related behaviour, but not pain‐related behaviour, was blocked by co‐administration of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant. Tissue levels of the endocannabinoids N‐arachidonoylethanolamide (anandamide, AEA) and 2‐AG were similar in the vHip of fear‐conditioned rats receiving formalin injection and the vHip of fear‐conditioned rats receiving saline injection. However, the levels of AEA and 2‐AG were significantly lower in the contralateral ventrolateral periaqueductal grey of formalin‐treated fear‐conditioned rats than in that of their saline‐treated counterparts. These data suggest that 2‐AG–CB1 receptor signalling in the vHip has an anti‐aversive effect, and that this effect is abolished in the presence of a persistent pain state.