Gemma K. Ford

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.

Clinical correlates of stress-induced analgesia: evidence from pharmacological studies

Exposure to aversive stimuli or contexts results in profound analgesia. Beecher [3] observed that World War II soldiers suffering from battle wounds often experienced little pain, whereas similar injuries in a non-threatening environment would be perceived as highly painful. These initial observations gave rise to the concept that the perception of pain is heavily influenced by context. The development of well-characterised, animal models of analgesia associated with aversion (unconditioned or conditioned stress-induced analgesia (SIA)), provided the first opportunity to study the neurobiological mechanisms underpinning this evolutionarily significant phenomenon. Clinical studies of SIA have substantiated evidence from animal studies and enhanced our knowledge of this phenomenon in humans. An increased understanding of the neurobiological mechanisms underpinning this extremely potent form of endogenous analgesia is of both fundamental physiological importance and potential therapeutic significance. Here we provide a brief initial overview and update of preclinical studies of SIA and then examine the extent to which the pharmacological mechanisms identified also apply in humans.

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.

Topical reviewClinical correlates of stress-induced analgesia: Evidence from pharmacological studies

Exposure to aversive stimuli or contexts results in profound analgesia. Beecher [3] observed that World War II soldiers suffering from battle wounds often experienced little pain, whereas similar injuries in a non-threatening environment would be perceived as highly painful. These initial observations gave rise to the concept that the perception of pain is heavily influenced by context. The development of well-characterised, animal models of analgesia associated with aversion (unconditioned or conditioned stress-induced analgesia (SIA)), provided the first opportunity to study the neurobiological mechanisms underpinning this evolutionarily significant phenomenon. Clinical studies of SIA have substantiated evidence from animal studies and enhanced our knowledge of this phenomenon in humans. An increased understanding of the neurobiological mechanisms underpinning this extremely potent form of endogenous analgesia is of both fundamental physiological importance and potential therapeutic significance. Here we provide a brief initial overview and update of preclinical studies of SIA and then examine the extent to which the pharmacological mechanisms identified also apply in humans.

Investigating the effects of distracting stimuli on nociceptive behaviour and associated alterations in brain monoamines in rats

Distraction interventions are used clinically to relieve pain. Exposure to distracting stimuli causes withdrawal of attention from the painful stimulus and reduces perceived pain. However, the neurobiological mechanisms mediating distraction‐induced analgesia are poorly understood due, in part, to a paucity of animal studies modelling this phenomenon. The present study investigated the effects of three distracting stimuli on formalin‐evoked nociceptive behaviour and brain tissue monoamine levels in rats. The three distractors were: exposure to a novel environment, exposure to a novel object, and exposure, without contact, to another rat (conspecific). A control group, habituated to the test arena, was also included. Formalin‐evoked nociceptive behaviour was significantly reduced in rats exposed to the novel object or novel arena, but not those exposed to the conspecific, compared with controls. Antinociception resulting from exposure to the novel object was of longer duration than that resulting from exposure to the novel arena. Failure to detect any distractor‐induced effects on plasma corticosterone levels or aversive behaviours suggests that the stimuli used were non‐stressful. HPLC analysis revealed that there was a significant reduction in serotonin and dopamine metabolites in the medial prefrontal cortex in animals exposed to the novel object. These results indicate that exposure to a novel object or arena reduces nociceptive behaviour in rats, effects accompanied by discrete alterations in serotonin and dopamine metabolites in the medial prefrontal cortex.

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.

Fear-induced suppression of nociceptive behaviour and activation of Akt signalling in the rat periaqueductal grey: role of fatty acid amide hydrolase

The endocannabinoid system regulates nociception and aversion and mediates fear-conditioned analgesia (FCA). We investigated the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which inhibits the catabolism of the endocannabinoid anandamide and related N-acylethanolamines, on expression of FCA and fear and pain related behaviour per se in rats. We also examined associated alterations in the expression of the signal transduction molecule phospho-Akt in the periaqueductal grey (PAG) by immunoblotting. FCA was modelled by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. URB597 (0.3 mg/kg, i.p.) enhanced FCA and increased fear-related behaviour in formalin-treated rats. Conditioned fear per se in non-formalin-treated rats was associated with increased expression of phospho-Akt in the PAG. URB597 reduced the expression of fear-related behaviour in the early part of the trial, an effect that was accompanied by attenuation of the fear-induced increase in phospho-Akt expression in the PAG. Intra-plantar injection of formalin also reduced the fear-induced increase in phospho-Akt expression. These data provide evidence for a role of FAAH in FCA, fear responding in the presence or absence of nociceptive tone, and fear-evoked increases in PAG phospho-Akt expression. In addition, the results suggest that fear-evoked activation of Akt signalling in the PAG is abolished in the presence of nociceptive tone.

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.

Impaired recognition memory and cognitive flexibility in the rat L5–L6 spinal nerve ligation model of neuropathic pain

Abstract Background and aims Although neuropathic pain is known to negatively affect cognition, the neural mechanisms involved are poorly understood. Chronic pain is associated with changes in synaptic plasticity in the brain which may impact on cognitive functioning. The aim of this study was to model neuropathic pain in mid-aged rats using spinal nerve ligation (SNL). Following establishment of allodynia and hyperalgesia, behaviour was assessed in a battery of cognitive tests. Expression of the presynaptic protein, synaptophysin, and its colocalisation with the vesicular GABA and glutamate transporters (vGAT and vGLUT, respectively), was investigated in the medial prefrontal cortex (mPFC) and hippocampus. Methods Nine month old male Sprague Dawley rats underwent L5-L6 spinal nerve ligation or a sham procedure. Mechanical and cold allodynia and thermal hyperalgesia were assessed using von Frey, acetone and Hargreaves tests, respectively. Cognition was assessed in the novel-object recognition, air-puff passive avoidance and Morris water maze behavioural tasks. Immunohistochemistry was used to examine the expression of synaptophysin in the mPFC and CA1 region of the hippocampus and double labelling of synaptophysin and the vesicular transporters vGAT and vGlut was used to investigate the distribution of synaptophysin on GABAergic and glutamatergic neurons. Results SNL rats displayed impaired performance in the novel-object recognition task. Passive-avoidance responding, and spatial learning and memory in the Morris water maze, were unaffected by SNL surgery. However, in the water maze reversal task, pain-related impairments were evident during training and probe trials. SNL surgery was not associated with any differences in the expression of synaptophysin or its colocalisation with vGAT or vGLUT in the mPFC or the hippocampal CA1 region. Conclusions These results suggest that the SNL model of neuropathic pain is associated with deficits in recognition memory and cognitive flexibility, but these deficits are not associated with altered synaptophysin expression or distribution in the mPFC and CA1. Implications Cognitive complaints are common amongst chronic pain patients. Here we modelled cognitive impairment in a well-established animal model of neuropathic pain and investigated the neural mechanisms involved. A better understanding of this phenomenon is an important prerequisite for the development of improved treatment of patients affected.

Behavioural, neurochemical and neuroendocrine effects of the endogenous β-carboline harmane in fear-conditioned rats

The putative endogenous imidazoline binding site ligand harmane enhances neuronal activation in response to psychological stress and alters behaviour in animal models of anxiety and antidepressant efficacy. However, the neurobiological mechanisms underlying harmane’s psychotropic effects are poorly understood. We investigated the effects of intraperitoneal injection of harmane (2.5 and 10 mg/kg) on fear-conditioned behaviour, hypothalamo-pituitary-adrenal axis activity, and monoaminergic activity within specific fear-associated areas of the rat brain. Harmane had no significant effect on the duration of contextually induced freezing or 22 kHz ultrasonic vocalisations and did not alter the contextually induced suppression of motor activity, including rearing. Harmane reduced the duration of rearing and tended to increase freezing in non-fear-conditioned controls, suggesting potential sedative effects. Harmane increased plasma ACTH and corticosterone concentrations, and serotonin (in hypothalamus, amygdaloid cortex, prefrontal cortex and hippocampus) and noradrenaline (prefrontal cortex) content, irrespective of fear-conditioning. Furthermore, harmane reduced dopamine and serotonin turnover in the PFC and hypothalamus, and serotonin turnover in the amygdaloid cortex in both fear-conditioned and non-fear-conditioned rats. In contrast, harmane increased dopamine and noradrenaline content and reduced dopamine turnover in the amygdala of fear-conditioned rats only, suggesting differential effects on catecholaminergic transmission in the presence and absence of fear. The precise mechanism(s) mediating these effects of harmane remain to be determined but may involve its inhibitory action on monoamine oxidases. These findings support a role for harmane as a neuromodulator, altering behaviour, brain neurochemistry and neuroendocrine function.