Effects of Intra-BLA Administration of PPAR Antagonists on Formalin-Evoked Nociceptive Behaviour, Fear-Conditioned Analgesia, and Conditioned Fear in the Presence or Absence of Nociceptive Tone in Rats.

There is evidence for the involvement of peroxisome proliferator-activated receptors (PPARs) in pain, cognition, and anxiety. However, their role in pain-fear interactions is unknown. The amygdala plays a key role in pain, conditioned fear, and fear-conditioned analgesia (FCA). We investigated the effects of intra-basolateral amygdala (BLA) administration of PPARα, PPARß/δ, and PPARγ antagonists on nociceptive behaviour, FCA, and conditioned fear in the presence or absence of nociceptive tone. Male Sprague-Dawley (SD) rats received footshock (FC) or no footshock (NFC) in a conditioning arena. Twenty-three and a half hours later, rats received an intraplantar injection of formalin or saline and, 15 min later, intra-BLA microinjections of vehicle, PPARα (GW6471) PPARß/δ (GSK0660), or PPARγ (GW9662) antagonists before arena re-exposure. Pain and fear-related behaviour were assessed, and neurotransmitters/endocannabinoids measured post-mortem. Intra-BLA administration of PPARα or PPARγ antagonists potentiated freezing in the presence of nociceptive tone. Blockade of all PPAR subtypes in the BLA increased freezing and BLA dopamine levels in NFC rats in the absence of nociceptive tone. Administration of intra-BLA PPARα and PPARγ antagonists increased levels of dopamine in the BLA compared with the vehicle-treated counterparts. In conclusion, PPARα and PPARγ in the BLA play a role in the expression or extinction of conditioned fear in the presence or absence of nociceptive tone.

Increasing Endocannabinoid Tone Alters Anxiety-Like and Stress Coping Behaviour in Female Rats Prenatally Exposed to Valproic Acid.

Given the sex differences evident in the prevalence of autism, there is an increased awareness of the importance of including females in autism research to determine sexual dimorphism and sex-specific treatments. Cannabinoids and endocannabinoid modulators have been proposed as potential novel treatments for autism-related symptoms; however, few studies to date have examined if these pharmacological agents elicit sex-specific effects. The aim of the present study was to use the valproic acid (VPA) model of autism to compare the behavioural responses of male and female rats and examine the effects of increasing endocannabinoid tone on the behavioural responses of VPA-exposed female rats. These data revealed that VPA-exposed male, but not female, rats exhibit reduced social responding in the three-chamber and olfactory habituation/dishabituation (OHD) test during adolescence. In comparison, VPA-exposed female, but not male, adolescent rats exhibited anxiety-like behaviour in the elevated plus maze (EPM) and open field test (OFT). In VPA-exposed female rats, increasing 2-AG levels augmented anxiety-like behaviour in the EPM and OFT, while increasing AEA levels reduced stress coping behaviour in the swim stress test. These data highlight sexual dimorphic behaviours in the VPA model and indicate that enhancing endocannabinoid levels may exacerbate negative affective behaviour in VPA-exposed females. Thus, considerations should be paid to the possible sex-specific effects of cannabinoids for the treatment of symptoms associated with autism.

Exacerbated LPS/GalN-Induced Liver Injury in the Stress-Sensitive Wistar Kyoto Rat Is Associated with Changes in the Endocannabinoid System.

Acute liver injury (ALI) is a highly destructive and potentially life-threatening condition, exacerbated by physical and psychological stress. The endocannabinoid system plays a key role in modulating stress and hepatic function. The aim of this study was to examine the development of acute liver injury in the genetically susceptible stress-sensitive Wistar-Kyoto (WKY) rat compared with normo-stress-sensitive Sprague Dawley (SD) rats, and associated changes in the endocannabinoid system. Administration of the hepatotoxin lipopolysaccharide/D-Galactosamine (LPS/GalN) resulted in marked liver injury in WKY, but not SD rats, with increased alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH) plasma levels, significant histopathological changes, increased hepatic pro-inflammatory cytokine expression and caspase-3 activity and expression and reduced Glutathione (GSH) activity. Furthermore, compared to SD controls, WKY rats display increased anandamide and 2-Arachidonoylglycerol levels concurrent with decreased expression of their metabolic enzymes and a decrease in cannabinoid (CB)1 receptor expression following LPS/GalN. CB1 antagonism with AM6545 or CB2 agonism with JWH133 did not alter LPS/GalN-induced liver injury in SD or WKY rats. These findings demonstrate exacerbation of acute liver injury induced by LPS/GalN in a stress-sensitive rat strain, with effects associated with alterations in the hepatic endocannabinoid system. Further studies are required to determine if the endocannabinoid system mediates or modulates the exacerbation of liver injury in this stress-sensitive rat strain.

Pharmacological Blockade of PPAR Isoforms Increases Conditioned Fear Responding in the Presence of Nociceptive Tone.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors with three isoforms (PPARα, PPARß/δ, PPARγ) and can regulate pain, anxiety, and cognition. However, their role in conditioned fear and pain-fear interactions has not yet been investigated. Here, we investigated the effects of systemically administered PPAR antagonists on formalin-evoked nociceptive behaviour, fear-conditioned analgesia (FCA), and conditioned fear in the presence of nociceptive tone in rats. Twenty-three and a half hours following fear conditioning to context, male Sprague-Dawley rats received an intraplantar injection of formalin and intraperitoneal administration of vehicle, PPARα (GW6471), PPARß/δ (GSK0660) or PPARγ (GW9662) antagonists, and 30 min later were re-exposed to the conditioning arena for 15 min. The PPAR antagonists did not alter nociceptive behaviour or fear-conditioned analgesia. The PPARα and PPARß/δ antagonists prolonged context-induced freezing in the presence of nociceptive tone without affecting its initial expression. The PPARγ antagonist potentiated freezing over the entire trial. In conclusion, pharmacological blockade of PPARα and PPARß/δ in the presence of formalin-evoked nociceptive tone, impaired short-term, within-trial fear-extinction in rats without affecting pain response, while blockade of PPARγ potentiated conditioned fear responding. These results suggest that endogenous signalling through these three PPAR isoforms may reduce the expression of conditioned fear in the presence of nociceptive tone.

Endocannabinoid modulation of inflammatory hyperalgesia in the IFN-α mouse model of depression.

Depression is a well-recognised effect of long-term treatment with interferon-alpha (IFN-α), a widely used treatment for chronic viral hepatitis and malignancy. In addition to the emotional disturbances, high incidences of painful symptoms such as headache and joint pain have also been reported following IFN-α treatment. The endocannabinoid system plays an important role in emotional and nociceptive processing, however it is unknown whether repeated IFN-α administration induces alterations in this system. The present study investigated nociceptive responding in the IFN-α-induced mouse model of depression and associated changes in the endocannabinoid system. Furthermore, the effects of modulating peripheral endocannabinoid tone on inflammatory pain-related behaviour in the IFN-α model was examined. Repeated IFN-α administration (8000 IU/g/day) to male C57/Bl6 mice increased immobility in the forced swim test and reduced sucrose preference, without altering body weight gain or locomotor activity, confirming development of the depressive-like phenotype. There was no effect of repeated IFN-α administration on latency to respond in the hot plate test on day 4 or 7 of treatment, however, formalin-evoked nociceptive behaviour was significantly increased in IFN-α treated mice following 8 days of IFN-α administration. 2-Arachidonoyl glycerol (2-AG) levels in the periaqueductal grey (PAG) and rostroventromedial medulla (RVM), and anandamide (AEA) levels in the RVM, were significantly increased in IFN-α-, but not saline-, treated mice following formalin administration. There was no change in endocannabinoid levels in the prefrontal cortex, spinal cord or paw tissue between saline- or IFNα-treated mice in the presence or absence of formalin. Furthermore, repeated IFN-α and/or formalin administration did not alter mRNA expression of genes encoding the endocannabinoid catabolic enzymes (fatty acid amide hydrolase or monoacylglycerol lipase) or endocannabinoid receptor targets (CB1, CB2 or PPARs) in the brain, spinal cord or paw tissue. Intra plantar administration of PF3845 (1 µg/10 µl) or MJN110 (1 µg/10 µl), inhibitors of AEA and 2-AG catabolism respectively, attenuated formalin-evoked hyperalgesia in IFN-α, but not saline-, treated mice. In summary, increasing peripheral endocannabinoid tone attenuates inflammatory hyperalgesia induced following repeated IFN-α administration. These data provide support for the endocannabinoid system in mediating and modulating heightened pain responding associated with IFNα-induced depression.

FAAH, but not MAGL, inhibition modulates acute TLR3-induced neuroimmune signaling in the rat, independent of sex.

Toll-like receptor (TLR)3 is a key component of the innate immune response to viral infection. The present study firstly examined whether sex differences exist in TLR3-induced inflammatory, endocrine, and sickness responses. The data revealed that TLR3-induced expression of interferon- or NFkB-inducible genes (IFN-α/ß, IP-10, or TNF-α), either peripherally (spleen) or centrally (hypothalamus), did not differ between male and female rats, with the exception of TLR3-induced IFN-α expression in the spleen of female, but not male, rats 8 hr post TLR3 activation. Furthermore, TLR3 activation increased plasma corticosterone levels, induced fever, and reduced locomotor activity and body weight - effects independent of sex. Thus, the acute-phase inflammatory, endocrine, and sickness responses to TLR3 activation exhibit minimal sex-related differences. A further aim of this study was to examine whether enhancing endocannabinoid tone - namely, 2-arachidonylglycerol (2-AG) or N-arachidonoylethanolamine (AEA), exhibited similar effects on TLR3-induced inflammatory responses in male versus female rats. Systemic administration of the monoacylglycerol lipase (MAGL) inhibitor MJN110 and subsequent increases in 2-AG levels did not alter the TLR3-induced increase in IP-10, IRF7, or TNF-α expression in the spleen or the hypothalamus of male or female rats. In contrast, the fatty acid amide hydrolase (FAAH) inhibitor URB597 increased levels of AEA and related N-acylethanolamines, an effect associated with the attenuation of TLR3-induced inflammatory responses in the hypothalamus, but not the spleen, of male and female rats. These data support a role for FAAH, but not MAGL, substrates in the modulation of TLR3-induced neuroinflammatory responses, effects independent of sex.

Psychological stress in early life as a predisposing factor for the development of chronic pain: Clinical and preclinical evidence and neurobiological mechanisms.

A wealth of research over the past 2 decades has expanded our understanding of the impact of early-life adversity on physiological function and, consequently, health and wellbeing in later life. Early-life adversity increases the risk of developing a number of disorders, such as chronic pain, fibromyalgia, and irritable bowel syndrome. Although much of the research has examined the impact of physical maltreatment, an increasing number of studies have been published over the past few years examining the effect of childhood psychological stress and trauma on the development of various types of chronic pain conditions. We review the clinical and preclinical data examining the link among early-life psychological stress, altered nociceptive behavior, and chronic pain in later life. Evidence supporting a role for certain key neurobiological substrates, including the hypothalamic-pituitary-adrenal axis; monoaminergic, opioidergic, endocannabinoid and immune systems; and epigenetic mechanisms in the association between early-life psychological stress and chronic pain, is provided. Greater understanding of the impact of early-life stress may inform the development of personalized treatments for chronic pain in later life and strategies to prevent its onset in susceptible individuals. © 2016 Wiley Periodicals, Inc.

Pharmacological inhibition of FAAH modulates TLR-induced neuroinflammation, but not sickness behaviour: An effect partially mediated by central TRPV1.

Aberrant activation of toll-like receptors (TLRs), key components of the innate immune system, has been proposed to underlie and exacerbate a range of central nervous system disorders. Increasing evidence supports a role for the endocannabinoid system in modulating inflammatory responses including those mediated by TLRs, and thus this system may provide an important treatment target for neuroinflammatory disorders. However, the effect of modulating endocannabinoid tone on TLR-induced neuroinflammation in vivo and associated behavioural changes is largely unknown. The present study examined the effect of inhibiting fatty acid amide hydrolyase (FAAH), the primary enzyme responsible for the metabolism of anandamide (AEA), in vivo on TLR4-induced neuroimmune and behavioural responses, and evaluated sites and mechanisms of action. Systemic administration of the FAAH inhibitor PF3845 increased levels of AEA, and related FAAH substrates N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA), in the frontal cortex and hippocampus of rats, an effect associated with an attenuation in the expression of pro- and anti-inflammatory cytokines and mediators measured 2hrs following systemic administration of the TLR4 agonist, lipopolysaccharide (LPS). These effects were mimicked by central i.c.v. administration of PF3845, but not systemic administration of the peripherally-restricted FAAH inhibitor URB937. Central antagonism of TRPV1 significantly attenuated the PF3845-induced decrease in IL-6 expression, effects not observed following antagonism of CB1, CB2, PPARα, PPARγ or GPR55. LPS-induced a robust sickness-like behavioural response and increased the expression of markers of glial activity and pro-inflammatory cytokines over 24hrs. Systemic administration of PF3845 modulated the TLR4-induced expression of neuroimmune mediators and anhedonia without altering acute sickness behaviour. Overall, these findings support an important role for FAAH substrates directly within the brain in the regulation of TLR4-associated neuroinflammation and highlight a role for TRPV1 in partially mediating these effects.

Pharmacological inhibition of fatty acid amide hydrolase attenuates social behavioural deficits in male rats prenatally exposed to valproic acid.

Autism spectrum disorders are a group of neurodevelopmental disorders characterised by impaired social interaction, deficits in communication and repetitive stereotyped behaviours. The endocannabinoid system plays an important role in modulating emotionality and social responding, however there have been a paucity of studies investigating this system in autistic animal models. This study investigated the effect of inhibiting fatty acid amide hydrolyase (FAAH), the anandamide catabolic enzyme, on behavioural responding in the valproic acid (VPA) rat model of autism. Male rats prenatally exposed to VPA exhibit an autistic-like behavioural phenotype exemplified as thermal hypoalgesia, reduced social and exploratory behaviour, and enhanced repetitive behaviour. Systemic administration of the FAAH inhibitor PF3845 (10mg/kg) attenuated the deficit in social behaviour observed in VPA exposed male animals without altering nociceptive, repetitive or exploratory behaviour. In comparison, female VPA exposed rats displayed enhanced repetitive and reduced exploratory behaviour, but no change in social behaviour or thermal nociceptive responding. PF3845 did not alter social, repetitive or thermal nociceptive responding, but reduced exploratory behaviour in a social context in VPA-, but not saline-, exposed females. These data indicate that FAAH inhibition elicits sexual dimorphic effects on behavioural responding in VPA exposed rodents, and support an important role for FAAH in the regulation of social behavioural deficits in autistic males.

Genotype-dependent responsivity to inflammatory pain: A role for TRPV1 in the periaqueductal grey.

Negative affective state has a significant impact on pain, and genetic background is an important moderating influence on this interaction. The Wistar-Kyoto (WKY) inbred rat strain exhibits a stress-hyperresponsive, anxiety/depressive-like phenotype and also displays a hyperalgesic response to noxious stimuli. Transient receptor potential subfamily V member 1 (TRPV1) within the midbrain periaqueductal grey (PAG) plays a key role in regulating both aversive and nociceptive behaviour. In the present study, we investigated the role of TRPV1 in the sub-columns of the PAG in formalin-evoked nociceptive behaviour in WKY versus Sprague-Dawley (SD) rats. TRPV1 mRNA expression was significantly lower in the dorsolateral (DL) PAG and higher in the lateral (L) PAG of WKY rats, compared with SD counterparts. There were no significant differences in TRPV1 mRNA expression in the ventrolateral (VL) PAG between the two strains. TRPV1 mRNA expression significantly decreased in the DLPAG and increased in the VLPAG of SD, but not WKY rats upon intra-plantar formalin administration. Intra-DLPAG administration of either the TRPV1 agonist capsaicin, or the TRPV1 antagonist 5'-Iodoresiniferatoxin (5'-IRTX), significantly increased formalin-evoked nociceptive behaviour in SD rats, but not in WKY rats. The effects of capsaicin were likely due to TRPV1 desensitisation, given their similarity to the effects of 5'-IRTX. Intra-VLPAG administration of capsaicin or 5'-IRTX reduced nociceptive behaviour in a moderate and transient manner in SD rats, and similar effects were seen with 5'-IRTX in WKY rats. Intra-LPAG administration of 5'-IRTX reduced nociceptive behaviour in a moderate and transient manner in SD rats, but not in WKY rats. These results indicate that modulation of inflammatory pain by TRPV1 in the PAG occurs in a sub-column-specific manner. The data also provide evidence for differences in the expression of TRPV1, and differences in the effects of pharmacological modulation of TRPV1 in specific PAG sub-columns, between WKY and SD rats, suggesting that TRPV1 expression and/or functionality in the PAG plays a role in hyper-responsivity to noxious stimuli in a genetic background prone to negative affect.

High Times for Painful Blues: The Endocannabinoid System in Pain-Depression Comorbidity.

Depression and pain are two of the most debilitating disorders worldwide and have an estimated cooccurrence of up to 80%. Comorbidity of these disorders is more difficult to treat, associated with significant disability and impaired health-related quality of life than either condition alone, resulting in enormous social and economic cost. Several neural substrates have been identified as potential mediators in the association between depression and pain, including neuroanatomical reorganization, monoamine and neurotrophin depletion, dysregulation of the hypothalamo-pituitary-adrenal axis, and neuroinflammation. However, the past decade has seen mounting evidence supporting a role for the endogenous cannabinoid (endocannabinoid) system in affective and nociceptive processing, and thus, alterations in this system may play a key role in reciprocal interactions between depression and pain. This review will provide an overview of the preclinical evidence supporting an interaction between depression and pain and the evidence supporting a role for the endocannabinoid system in this interaction.

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.

Minocycline modulates neuropathic pain behaviour and cortical M1-M2 microglial gene expression in a rat model of depression.

There is a paucity of data on the role of microglia and neuroinflammatory processes in the association between chronic pain and depression. The current study examined the effect of the microglial inhibitor minocycline on depressive-like behaviour, spinal nerve ligation (SNL)-induced mechanical and cold allodynia and associated changes in the expression of genes encoding microglial markers (M1 vs. M2 polarisation) and inflammatory mediators in the prefrontal cortex in the olfactory bulbectomised (OB) rat model of depression. Acute minocycline administration did not alter OB-induced depressive-like behaviour but prevented SNL-induced mechanical allodynia in both OB and sham rats. In comparison, chronic minocycline attenuated OB-induced depressive-like behaviour and prevented the development of SNL-induced mechanical allodynia in OB, but not sham, rats. Further analysis revealed that SNL-induced mechanical allodynia in OB rats was attenuated by chronic minocycline at almost all time-points over a 2week testing period, an effect observed only from day 10 post-SNL in sham rats. Chronic administration of minocycline reduced the expression of CD11b, a marker of microglial activation, and the M1 pro-inflammatory cytokine IL-1ß, in the prefrontal cortex of sham-SNL animals. In comparison, the expression of the M2 microglia marker (MRC2) and anti-inflammatory cytokine IL-10 was increased, as were IL-1ß, IL-6 and SOCS3, in the prefrontal cortex of OB-SNL animals following chronic minocycline. Thus, chronic minocycline attenuates neuropathic pain behaviour and modulates microglial activation and the central expression of inflammatory mediators in a manner dependent on the presence or absence of a depressive-like phenotype.

Surveillance of pathogenic bacteria on a food matrix using machine-learning-enabled paper chromogenic arrays.

Global food systems can benefit significantly from continuous monitoring of microbial food safety, a task for which tedious operations, destructive sampling, and the inability to monitor multiple pathogens remain challenging. This study reports significant improvements to a paper chromogenic array sensor - machine learning (PCA-ML) methodology sensing concentrations of volatile organic compounds (VOCs) emitted on a species-specific basis by pathogens by streamlining dye selection, sensor fabrication, database construction, and machine learning and validation. This approach enables noncontact, time-dependent, simultaneous monitoring of multiple pathogens (Listeria monocytogenes, Salmonella, and E. coli O157:H7) at levels as low as 1 log CFU/g with over 90% accuracy. The report provides theoretical and practical frameworks demonstrating that chromogenic response, including limits of detection, depends on time integrals of VOC concentrations. The paper also discusses the potential for implementing PCA-ML in the food supply chain for different food matrices and pathogens, with species- and strain-specific identification.

Characterization of pain-, anxiety-, and cognition-related behaviors in the complete Freund's adjuvant model of chronic inflammatory pain in Wistar-Kyoto rats.

Introduction: Chronic pain is often associated with comorbid anxiety and cognitive dysfunction, negatively affecting therapeutic outcomes. The influence of genetic background on such interactions is poorly understood. The stress-hyperresponsive Wistar-Kyoto (WKY) rat strain, which models aspects of anxiety and depression, displays enhanced sensitivity to noxious stimuli and impaired cognitive function, compared with Sprague-Dawley (SD) counterparts. However, pain- and anxiety-related behaviors and cognitive impairment following induction of a persistent inflammatory state have not been investigated simultaneously in the WKY rats. Here we compared the effects of complete Freund's adjuvant (CFA)-induced persistent inflammation on pain-, negative affect- and cognition-related behaviors in WKY vs. SD rats. Methods: Male WKY and SD rats received intra-plantar injection of CFA or needle insertion (control) and, over the subsequent 4 weeks, underwent behavioral tests to assess mechanical and heat hypersensitivity, the aversive component of pain, and anxiety- and cognition-related behaviors. Results: The CFA-injected WKY rats exhibited greater mechanical but similar heat hypersensitivity compared to SD counterparts. Neither strain displayed CFA-induced pain avoidance or anxiety-related behavior. No CFA-induced impairment was observed in social interaction or spatial memory in WKY or SD rats in the three-chamber sociability and T-maze tests, respectively, although strain differences were apparent. Reduced novel object exploration time was observed in CFA-injected SD, but not WKY, rats. However, CFA injection did not affect object recognition memory in either strain. Conclusions: These data indicate exacerbated baseline and CFA-induced mechanical hypersensitivity, and impairments in novel object exploration, and social and spatial memory in WKY vs. SD rats.

Stress-induced changes in nociceptive responding post-surgery in preclinical rodent models.

Chronic post-surgical pain affects up to 85% of individuals depending on the type of surgery, the extent of inflammation, tissue and/or nerve damage. Pre-surgical stress is associated with greater pain intensity, prolonged recovery and is one of the main risk factors for the development of chronic post-surgical pain. Clinically valid animal models provide an important means of examining the mechanisms underlying the effects of stress on post-surgical pain and identifying potential novel therapeutic targets. This review discusses the current data from preclinical animal studies examining the effect of stress on post-surgical pain, the potential underlying mechanisms and gaps in the knowledge that require further investigation.

Sexually Dimorphic Expression of Fear-conditioned Analgesia in Rats and Associated Alterations in the Endocannabinoid System in the Periaqueductal Grey.

The endocannabinoid system within the periaqueductal grey (PAG) has been implicated in fear-conditioned analgesia (FCA), the profound suppression of pain upon re-exposure to a context previously paired with an aversive stimulus. Since the endocannabinoid and nociceptive systems exhibit sexual dimorphism, the aim of the present study was to assess possible sex differences in the expression of FCA, fear in the presence of nociceptive tone, and associated sex-dependent alterations in the endocannabinoid system within the PAG. Male and female Sprague-Dawley rats received footshock (10 × 1s; 0.4 mA; every 60 s) or no-footshock in a conditioning arena and 23.5 h later received intraplantar injection of formalin (2.5%) under brief isoflourane anaesthetic into the right hind paw. Nociceptive and fear-related behaviours were assessed 30 min later. Levels of endocannabinoids, N-acylethanolamines and neurotransmitters in the PAG were assessed by LC-MS/MS and expression of endocannabinoid system-related proteins by Western immunoblotting. Male, but not female, rats exhibited robust FCA and greater expression of fear-related behaviours than females. Fear-conditioned formalin-treated males, but not females, had higher levels of N-oleoylethanolamine (OEA) and γ-aminobutyric acid (GABA) in the PAG, compared with non-fear-conditioned controls. There was no effect of fear conditioning on the levels of FAAH or CB1 receptor expression (CB1R) in the PAG of male or female formalin-treated rats. Non-fear-conditioned females had higher levels of CB1R and PPARγ expression than non-fear-conditioned male counterparts. In summary, our results provide evidence of sexual dimorphism in the expression of FCA and fear-related behaviours, and associated alterations in components of the endocannabinoid system and GABA within the PAG.

Exploring Parental Responses to Pre-schoolers' "Everyday" Pain Experiences Through Electronic Diary and Ecological Momentary Assessment Methodologies.

Objective: Parental influence during children's "everyday" pain events is under-explored, compared to clinical or experimental pains. We trialed two digital reporting methods for parents to record the real-world context surrounding their child's everyday pain events within the family home. Methods: Parents (N = 21) completed a structured e-diary for 14 days, reporting on one pain event experienced by their child (aged 2.5-6 years) each day, and describing child pain responses, parental supervision, parental estimates of pain severity and intensity, and parental catastrophizing, distress, and behavioral responses. During the same 2-week period, a subsample of parent-child pairs (N = 9) completed digital ecological momentary assessments (EMA), immediately after any chosen pain event. Children reported their current pain while parents estimated the child's pain and indicated their own distress. Results: "Everyday" pain events frequently featured minor injuries to the child's head, hands or knees, and child responses included crying and non-verbal comments (e.g., "Ouch!"). Pain events occurred less frequently when parents had been supervising their child, and supervising parents reported lower levels of worry and anxiety than non-supervising parents. Child sex was significantly associated with parental estimates of pain intensity, with parents of girls giving higher estimates than parents of boys. Child age was significantly associated with both the number of pain events and with parental estimates of pain intensity and child distress: the youngest children (2-3 years) experienced the fewest pain events but received higher pain and distress estimates from parents than older children. Hierarchal Linear Modeling revealed that parental estimates of pain severity were significant positive predictors of parental distress and catastrophizing in response to a specific pain event. Furthermore, higher levels of parental catastrophic thinking in response to a specific pain event resulted in increased distress, solicitousness, and coping-promoting behaviors in parents. The EMA data revealed that children reported significantly higher pain intensity than their parents. Conclusion: The electronic pain diary provided a key insight into the nature of "everyday" pain experiences around the family home. Digital daily reporting of how the family copes with "everyday" events represents a viable means to explore a child's everyday pains without disrupting their home environment.

Pharmacological Blockade of PPARα Exacerbates Inflammatory Pain-Related Impairment of Spatial Memory in Rats.

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that exist in three isoforms: PPARα, PPARß/δ and PPARγ. Studies suggest that the PPAR signalling system may modulate pain, anxiety and cognition. The aim of the present study was to investigate whether endogenous signalling via PPARs differentially modulates innate anxiety responses and mnemonic function in the presence and absence of inflammatory pain. We examined the effects of intraperitoneal administration of GW6471 (PPARα antagonist), GSK0660 (PPARß/δ antagonist), GW9662 (PPARγ antagonist), and N-palmitoylethanolamide (PEA) on rat behaviour in the elevated plus maze (EPM), open field (OF), light-dark box (LDB), and novel object recognition (NOR) tests in the presence or absence of chronic inflammatory pain. Complete Freund's Adjuvant (CFA)-injected rats exhibited impaired recognition and spatial mnemonic performance in the NOR test and pharmacological blockade of PPARα further impaired spatial memory in CFA-treated rats. N-oleoylethanolamide (OEA) levels were higher in the dorsal hippocampus in CFA-injected animals compared to their counterparts. The results suggest a modulatory effect of CFA-induced chronic inflammatory pain on cognitive processing, but not on innate anxiety-related responses. Increased OEA-PPARα signalling may act as a compensatory mechanism to preserve spatial memory function following CFA injection.

Mu-opioid receptor agonism differentially alters social behaviour and immediate early gene expression in male adolescent rats prenatally exposed to valproic acid versus controls.

Mu-opioid receptors (MOPs) mediate and modulate social reward and social interaction. However, few studies have examined the functionality of this system in rodent models of social impairment. Deficits in social motivation and cognition are observed in rodents following pre-natal exposure to the anti-epileptic valproic acid (VPA), however it is not known whether MOP functionality is altered in these animals. The present study examined the effects of acute administration of the prototypical MOP agonist morphine (1 mg/kg) on social behavioural responding in the 3-chamber test and immediate early gene expression in adolescent rats (postnatal day 28-43) prenatally exposed to VPA vs saline-exposed controls. Pharmacokinetic analysis of morphine concentration, MOP binding and expression were also examined. The data revealed that sociability and social novelty preference in the 3-chamber test were reduced in rats prenatally exposed to VPA compared to saline-exposed control counterparts. Morphine reduced both sociability and social novelty preference behaviour in saline-, but not VPA-, exposed rats. Analysis of immediate early gene expression revealed that morphine reduced the expression of cfos in the prefrontal cortex of both saline- and VPA-exposed rats and reduced expression of cfos and junb in the hippocampus of VPA-exposed rats only. Pharmacokinetic analysis revealed similar concentrations of morphine in the plasma and brain of both saline- and VPA-exposed rats and similar thalamic MOP occupancy levels. Gene and protein expression of MOP in prefrontal cortex and hippocampus did not differ between saline and VPA-exposed rats. These data indicate differential effects of morphine on social responding and immediate early gene expression in the hippocampus of VPA-exposed rats compared with saline-exposed controls. This study provides support for altered MOP functionality in rats prenatally exposed to VPA, which may underlie the social deficits observed in the model.