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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2018 Jul 14;176(10):1492–1505. doi: 10.1111/bph.14376

The prefrontal cortical endocannabinoid system modulates fear–pain interactions in a subregion‐specific manner

Kieran Rea 1,3, Fiona McGowan 1,3,†, Louise Corcoran 1,3,†, Michelle Roche 2,3, David P Finn 1,3,✉
PMCID: PMC6487600  PMID: 29847859

Abstract

Background and Purpose

The emotional processing and coordination of top‐down responses to noxious and conditioned aversive stimuli involves the medial prefrontal cortex (mPFC). Evidence suggests that subregions of the mPFC [infralimbic (IfL), prelimbic (PrL) and anterior cingulate (ACC) cortices] differentially alter the expression of contextually induced fear and nociceptive behaviour. We investigated the role of the endocannabinoid system in the IfL, PrL and ACC in formalin‐evoked nociceptive behaviour, fear‐conditioned analgesia (FCA) and conditioned fear in the presence of nociceptive tone.

Experimental Approach

FCA was modelled in male Lister‐hooded rats by assessing formalin‐evoked nociceptive behaviour in an arena previously paired with footshock. The effects of intra‐mPFC administration of AM251 [cannabinoid type 1 (CB1) receptor antagonist/inverse agonist], URB597 [fatty acid amide hydrolase (FAAH) inhibitor] or URB597 + AM251 on FCA and freezing behaviour were assessed.

Key Results

AM251 attenuated FCA when injected into the IfL or PrL and reduced contextually induced freezing behaviour when injected intra‐IfL but not intra‐PrL or intra‐ACC. Intra‐ACC administration of AM251 alone or in combination with URB597 had no effect on FCA or freezing. URB597 attenuated FCA and freezing behaviour when injected intra‐IfL, prolonged the expression of FCA when injected intra‐PrL and had no effect on these behaviours when injected intra‐ACC.

Conclusions and Implications

These results suggest important and differing roles for FAAH substrates or CB1 receptors in the PrL, IfL and ACC in the expression of FCA and conditioned fear in the presence of nociceptive tone.

Linked Articles

This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc

Abbreviations

ACC

anterior cingulate cortex

AM251

1‐(2,4‐dichlorophenyl)‐5‐(4‐iodophenyl)‐4‐methyl‐N‐(piperidin‐1‐yl)‐1H‐pyrazole‐3‐carboxamide

CPS

composite pain score

FAAH

fatty acid amide hydrolase

FC

fear‐conditioned

FCA

fear‐conditioned analgesia

IfL

infralimbic

mPFC

medial prefrontal cortex

NFC

non‐fear‐conditioned

PrL

prelimbic

URB597

cyclohexyl‐carbamic acid 3′‐carbamoyl‐biphenyl‐3‐yl ester

VEH

vehicle

Introduction

The medial prefrontal cortex (mPFC) is strongly involved in cognitive, emotional and motivational processes and in regulation of responses to aversion (Laviolette et al., 2005; Resstel et al., 2006; Wang et al., 2009; Gilmartin et al., 2013; Gilmartin et al., 2014; Jiang et al., 2014) and pain (Baulmann et al., 2000; Luongo et al., 2013; Okine et al., 2016). The mPFC comprises a number of subregions that can be differentiated by anatomical connectivity, cytoarchitecture and function. In rodents, the infralimbic (IfL) and prelimbic (PrL) subregions of the mPFC have been shown to differentially affect acquisition, consolidation and expression of contextually conditioned fear (Vidal‐Gonzalez et al., 2006; Corcoran and Quirk, 2007; Sierra‐Mercado et al., 2011; Sharpe and Killcross, 2014; Almada et al., 2015). Roles for the PrL in fear‐induced antinociception (Freitas et al., 2013) and formalin‐induced conditioned place avoidance (Jiang et al., 2014) have also been demonstrated. The anterior cingulate cortex (ACC) is a subregion of the mPFC involved in the modulation of fear behaviour (Einarsson et al., 2015), cognitive‐affective component of pain (Johansen et al., 2001) and in top‐down descending modulation of pain (Fuchs et al., 2014). The ACC is connected reciprocally with both the PrL and the IfL and may play a role in modulating their output (Vertes, 2002). Despite the evidence for a role of the IfL, PrL and ACC in fear‐related and pain‐related behaviour, there is a paucity of studies comparing these three subregions of the mPFC with respect to their role in fear–pain interactions, which we sought to address here.

Specifically, we investigated the role of the endogenous cannabinoid (endocannabinoid) system within subregions of the mPFC in the expression of formalin‐evoked nociceptive tone, fear‐conditioned analgesia (FCA) and conditioned fear in the presence of nociceptive tone. The endocannabinoid system consists of cannabinoid CB1 (Devane et al., 1988; Matsuda et al., 1990) and CB2 (Munro et al., 1993) receptors, their endogenous ligands (or endocannabinoids), the two best characterized being N‐arachidonoylethanolamide [anandamide (AEA)] and 2‐arachidonoyl glycerol (Devane et al., 1992; Mechoulam et al., 1995; Sugiura et al., 1995), and the enzymes responsible for the synthesis and degradation of the endocannabinoids. FCA is the robust suppression of nociceptive behaviour during or following expression of classical Pavlovian conditioned fear (Ford and Finn, 2008; Butler and Finn, 2009). Our previous research has implicated the PFC in the expression of FCA in rats (Butler et al., 2011). Moreover, we and others have demonstrated a key role for the endocannabinoid system in FCA (Finn et al., 2003; Finn et al., 2004; Roche et al., 2007; Butler et al., 2008; Ford and Finn, 2008; Butler and Finn, 2009; Ford et al., 2011; Olango et al., 2012; Rea et al., 2013; Olango et al., 2014; Corcoran et al., 2015) and unconditioned stress‐induced analgesia (Hohmann et al., 2005; Suplita et al., 2005; Connell et al., 2006; Guindon and Hohmann, 2009). These studies have highlighted the importance of the endocannabinoid system in discrete brain regions including the amygdala (Rea et al., 2013), hippocampus (Ford et al., 2011) and periaqueductal grey (Olango et al., 2012), all of which are connected anatomically to the mPFC. Components of the endocannabinoid system, including CB1 receptors and the anandamide‐catabolizing enzyme fatty acid amide hydrolase (FAAH), are highly expressed in the mPFC (Herkenham et al., 1991; Mailleux and Vanderhaeghen, 1992; Tsou et al., 1998; Egertova et al., 2003). Interestingly, Freitas et al. (2013) have demonstrated that innate, unconditioned fear‐induced antinociception arising from blockade of GABAA receptors in the ventromedial and dorsomedial hypothalamus is attenuated by microinjection of the CB1 receptor antagonist/inverse agonist AM251 into the PrL. However, no studies to date have compared the role of the endocannabinoid system in the PrL, IfL and ACC in pain suppression arising from conditioned fear (FCA), and this was the primary aim of the present study.

We tested the hypothesis that the endocannabinoid system in the PrL, IfL and ACC differentially modulates FCA and expression of fear in the presence of formalin‐evoked nociceptive tone. To this end, we investigated the effects of local intra‐PrL, intra‐IfL and intra‐ACC microinjections of the FAAH inhibitor, URB597 and the CB1 receptor antagonist/inverse agonist, AM251, alone or in combination, on formalin‐evoked nociceptive behaviour, FCA and expression of fear in the presence of formalin‐evoked nociceptive tone in rats. Elucidation of the role of the endocannabinoid system in different subregions of the mPFC in fear–pain interactions may facilitate increased understanding and improved treatment of pain‐related and fear‐related disorders and their comorbidity.

Methods

Animals

All animal care and experimental procedures were approved by the Animal Care and Research Ethics Committee, National University of Ireland, Galway, and the work carried out under licence from the Irish Department of Health and Children, in compliance with the European Communities Council directives 86/609 and 2010/63 and conformed to the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). A total of 280 male Lister‐hooded rats (260–350 g on day of behavioural testing; Charles River, Margate, Kent, UK) were used. Animals were housed 3–4 per cage before surgery and singly thereafter in plastic bottomed cages (L: 45 × H: 20 × W: 20 cm) with wood shavings as bedding. They were maintained at a constant temperature (22 ± 2°C) under standard lighting conditions (12:12 h light–dark, lights on from 0800 to 2000 h). Experiments were carried out during the light phase between 0800 and 1700 h. Food (14% Harlan‐Teklad‐2014 Maintenance Diet, Harlan Laboratories, Belton, Loughborough, UK) and water were available ad libitum.

Cannulae implantation

Animals were left to acclimatize for 4–8 days after delivery before surgery. Stainless steel guide cannulae (5 mm length, 22G; Plastics One Inc., Roanoke, VA, USA) were stereotaxically implanted bilaterally 1 mm above the right and left IfL [anteroposterior (AP) + 2 mm relative to bregma, medial lateral (ML) ± 1.5 mm relative to bregma and at a 12o angle and dorsal ventral (DV) – 3.6 mm from dura, toothbar set at −3 mm], or PrL (AP + 2.4 mm relative to bregma, ML ± 1.5 mm relative to bregma and at a 12o angle and DV – 2.3 mm from dura, toothbar set at −3 mm) or ACC (AP + 1 mm relative to bregma, ML ± 1.3 mm at a 12o angle and DV – 1.3 mm from dura, toothbar set at −3 mm) (Paxinos and Watson, 1998) under isoflurane anaesthesia (2–3% in O2; 0.5 L·min−1). Rats were deemed to be sufficiently anaesthetized when the pedal withdrawal reflex was absent. The cannulae were permanently fixed to the skull using stainless steel screws and carboxylate cement. A stylet made from stainless steel tubing (Plastics One Inc.; 0.356 mm diameter) was inserted into the guide cannula to prevent blockage by debris. The non‐steroidal anti‐inflammatory agent, carprofen (5 mg·kg−1 s.c.; Rimadyl; Pfizer, Kent, UK), and the broad spectrum antibiotic, enrofloxacin (2.5 mg·kg−1 s.c.; Baytril; Bayer Ltd., Dublin, Ireland), were administered before surgery to manage post‐operative pain and to prevent infection respectively. Following cannulae implantation, the rats were housed singly and administered enrofloxacin (2.5 mg·kg−1 s.c.) for a further 3 days. Rats were allowed to recover for at least 6 days prior to experimentation. During this period, the rats were handled daily, stylets checked and their body weight and general health monitored on a daily basis.

Drug preparation

On test days, stock solutions of 4 mM AM251 and 0.2 mM URB597 were prepared in 100% DMSO vehicle (VEH). For bilateral intra‐PrL, intra‐IfL and intra‐ACC microinjections, AM251 and URB597 were diluted to a concentration of 2 and 0.1 mM in 100% DMSO VEH, respectively, while the combination was prepared by adding equal volumes of the stock 4 mM AM251 and 0.2 mM URB597. These doses of URB597 and AM251 are based on previous work carried out by our laboratory and evidence from the literature in rat models of pain and fear (Laviolette and Grace, 2006; de Novellis et al., 2008; Lisboa et al., 2010; Ebrahimzadeh and Haghparast, 2011; Ford et al., 2011; Olango et al., 2012; Freitas et al., 2013; Rea et al., 2014a). A solution of 2.5% formalin (Sigma‐Aldrich) was prepared from a 37% stock solution diluted with 0.9% sterile saline.

Experimental procedures

The FCA paradigm was essentially as described previously (Finn et al., 2004; Roche et al., 2007; Butler et al., 2008; Rea et al., 2009). In brief, it consisted of two phases, conditioning and testing, occurring 24 h apart. On the conditioning day, rats were placed in a Perspex fear‐conditioning/observation chamber (30 × 30 × 30 cm), and after 15 s, they received the first of 10 footshocks (0.4 mA, 1 s duration; LE85XCT Programmer and Scrambled Shock Generator; Linton Instrumentation, Norfolk, UK) spaced 60 s apart. Fifteen seconds after the last footshock, rats were returned to their home cage. Controls not receiving footshock were exposed to the chamber for an equivalent 9.5 min period. Three experiments, all using a different cohort of rats, were carried out involving microinjections of URB597, AM251, URB597 + AM251 (combination) or VEH into the IfL, PrL or ACC respectively (Figure 1). The conditioning phase for these experiments was carried out as outlined above. The test phase commenced 23.5 h later when the rats received an intraplantar injection of 50 μL formalin (2.5% formaldehyde solution prepared in sterile saline) into the right hindpaw under brief isoflurane anaesthesia (2–3% in O2; 0.5 L·min−1). Fifteen minutes post‐formalin injection, rats received bilateral intra‐IfL, intra‐PrL or intra‐ACC microinjections of VEH (100% DMSO), 2 mM AM251, 0.1 mM URB597 or the combination of 2 mM AM251 with 0.1 mM URB597 in an injection volume of 0.3 μL over a 60 s time interval using an injection pump, a 1 μL Hamilton syringe and polyethylene tubing connected to a stainless steel injector with 1 mm protrusion beyond the guide cannula (28G; Plastics One Inc.). Immediately following the intracerebral microinjections, rats were returned to their home cages for 15 min prior to being placed into the same Perspex arenas in which they had been conditioned. A video camera located beneath the observation chamber was used to monitor animal behaviour. The video feed was recorded onto DVD for 30 min. The 30–60 min post‐formalin interval was chosen on the basis of previous studies demonstrating that formalin‐evoked nociceptive behaviour is stable over this time period, is endocannabinoid mediated and is subject to supraspinal modulation (Finn et al., 2004; Ford et al., 2011; Olango et al., 2012; Rea et al., 2011; Roche et al., 2010).

Figure 1.

Figure 1

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Diagram of the experimental paradigm, treatment groups and timeline.

At the end of the test phase (60 min post‐formalin injection), rats were killed by decapitation, and an intracerebral microinjection of Fastgreen dye (0.5 μL of 1% solution) was given for subsequent histological confirmation of the microinjection sites. The brains were then removed, snap‐frozen on dry ice and stored at −80°C.

Formalin‐induced oedema was assessed by measuring the change in the diameter of the right hindpaw immediately before, and 60 min after, formalin administration, using Vernier callipers.

This design resulted in eight experimental groups (starting n = 11–12 per group for surgery; final n after removal of outliers where the cannula placements were inaccurate or injections were suboptimal are shown in Table 1).

Table 1.

Summary of experimental groups and final n number per group in experiments 1 (IfL), 2 (PrL) and 3 (ACC)

Group Conditioning Formalin i.pl. Drug/VEH IfL (n) PrL (n) ACC (n)
1 FC Formalin 100% DMSO 5 9 12
2 No FC Formalin 100% DMSO 9 8 8
3 FC Formalin 2 mM AM251 6 8 9
4 No FC Formalin 2 mM AM251 9 8 11
5 FC Formalin 0.1 mM URB597 9 10 8
6 No FC Formalin 0.1 mM URB597 8 8 9
7 FC Formalin URB597 + AM251 5 9 8
8 No FC Formalin URB597 + AM251 8 8 7
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i.pl., intraplantar.

Behavioural analysis

Ethovision XT 7.0 software package (Noldus, Wageningen, The Netherlands) was used to analyse behaviour, allowing for continuous event recording over each 30 min trial. The behaviours assessed (by an experimenter blind to treatment) were duration of freezing (defined as the cessation of all visible movement except that necessary for breathing) as a measure of fear‐related behaviour and nociceptive behaviours [composite pain score (CPS)] as described previously (Finn et al., 2004; Finn et al., 2006; Butler et al., 2008). Nociceptive behaviours were measured using the weighted composite pain scoring technique (Watson et al., 1997). Nociceptive behaviours are divided into two types according to this method; the first (pain 1) is the time spent elevating the formalin‐injected paw without contact with any other surface. The second (pain 2) is the time spent holding, licking, biting, shaking or flinching the formalin‐injected paw. The CPS equation is given as composite pain score [CPS = (duration of pain 1 + 2 × duration of pain 2)∕(total duration of analysis period)] (Watson et al., 1997). The EthoVision system automatically tracked total distance moved as a measure of locomotor activity.

Histological verification of intracerebral microinjection sites

The sites of intracerebral microinjection were determined prior to data analysis. Brain sections (30 μm thickness) marked with Fastgreen dye mark were collected using a cryostat (Microm, Thermo Fisher Scientific, Walldorf, Germany), mounted on gelatinized glass slides and counterstained with cresyl violet to locate the precise position of microinjection sites under light microscopy.

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018). Rats were randomly assigned to experimental groups, and the sequence of testing was randomized throughout the experiment. Previous published studies and power analysis suggested that when using ANOVA, the sample sizes used would yield sufficient power to reliably detect differences in the means between groups with sufficient power (i.e. >90%). Results are expressed as group means ± SEM. The IBM SPSS statistical software package (SPSS v23.0 for Microsoft Windows, Chicago, IL, USA) was used to analyse all data. Normality and homogeneity of variance were assessed using Shapiro–Wilk's and Levene's test respectively. Paw oedema data were analysed using a paired Student's t‐test. Time course behavioural data were analysed by two‐way repeated measures ANOVA with time as the within‐subjects factor and fear conditioning and drug treatment as the between‐subjects factors. Post hoc pairwise comparisons were made with Tukey's test when appropriate. Differences between group means were considered significant when P < 0.05.

Materials

Formalin (37% formaldehyde solution), DMSO and URB597 were purchased from Sigma‐Aldrich, Dublin, Ireland. AM251 was purchased from Abcam plc, Cambridge, UK.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a,b).

Results

Histological verification of microinjection sites

After histological verification, 75% of the microinjections were found to be within the borders of both the left and the right IfL, 84% in the PrL and 78% in the ACC. The remaining microinjections were placed in the corpus callosum, or outside the borders of the IfL, PrL or ACC respectively. Only data from rats where intracerebral injections were accurately placed in both the left and the right IfL, PrL or ACC have been included in this paper (Figures 2A, B, 3A, B and 4A, B).

Figure 2.

Figure 2

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(A, B) Diagram of the confirmed microinjection sites of all animals with guide cannulae in the left and right IfL.

Figure 3.

Figure 3

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(A, B) Diagram of the confirmed microinjection sites of all animals that underwent surgery to place guide cannulae in the left and right PrL.

Figure 4.

Figure 4

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(A, B) Diagram of the confirmed microinjection sites of all animals that underwent surgery to place guide cannulae in the left and right ACC.

Effects of intra‐mPFC administration of AM251, URB597 or URB597 + AM251 on formalin‐evoked nociceptive behaviour and FCA

Intraplantar injection of formalin increased right hindpaw diameter (indicative of oedema) in each of the three studies (IfL PrL and ACC), and produced robust licking, biting, shaking, flinching and elevation of the injected right hindpaw.

In the IfL study, two‐way repeated measures ANOVA revealed a significant effect of fear conditioning, treatment and fear conditioning * treatment on CPS over the course of the 30 min testing period (Figure 5A). There was also a significant effect of time, time * fear conditioning, time * treatment and time * fear conditioning * treatment . Further post hoc analysis with Tukey's test revealed that fear‐conditioned (FC) VEH‐treated rats displayed significantly less formalin‐evoked nociceptive behaviour compared with non‐fear‐conditioned (NFC) VEH‐treated rats in the first 10 min of the testing period, confirming the expression of FCA. This FCA was significantly attenuated by intra‐IfL administration of either AM251 or URB597 alone and a non‐significant trend for a similar attenuation when both drugs were co‐administered. Intra‐IfL administration of these drugs had no significant effect on formalin‐evoked nociceptive behaviour in NFC rats (Figure 5A).

Figure 5.

Figure 5

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(A) Effects of fear conditioning and bilateral administration of URB597, AM251 or URB597 + AM251 directly into the IfL on formalin‐evoked nociceptive behaviour in rats over the full 30 min testing period subdivided into 10 min time bins. (B) Effects of fear conditioning and bilateral administration of URB597, AM251 or URB597 + AM251 directly into the IfL on the duration of freezing in formalin‐injected rats over the full 30 min testing period, subdivided into 10 min time bins. (C) Effects of fear conditioning and bilateral administration of URB597, AM251 or URB597 + AM251 directly into the IfL on the distance moved (cm) in formalin‐injected rats over the full 30 min testing period, subdivided into 10 min time bins. All data are expressed as mean ± SEM. *P < 0.05, significantly different from NFC; # P < 0.05, significantly different from FC VEH.

In the PrL study, similar analysis revealed a significant effect of fear conditioning but not treatment or fear conditioning * treatment on CPS over the course of the 30 min testing period (Figure 6A). There was also a significant effect of time and time * fear conditioning but not time * treatment or time * fear conditioning * treatment. Further post hoc analysis with Tukey's test revealed that FC VEH‐treated rats displayed significantly less formalin‐evoked nociceptive behaviour compared with NFC VEH‐treated rats in the first 10 min of the testing period, confirming the expression of FCA. This FCA was significantly attenuated by intra‐PrL administration of AM251 but not by URB597 or co‐administration of URB597 and AM251. Indeed, FC rats that received intra‐PrL URB597 (but not URB597 + AM251) had significantly lower formalin‐evoked nociceptive behaviour than NFC counterparts over the first 20 min of the trial, suggesting that URB597 prolonged the expression of FCA relative to VEH‐treated FC rats in which significant FCA was observed in the first 10 min only. This URB597‐induced prolongation of FCA was not observed in rats co‐treated with URB597 and AM251. Intra‐PrL administration of these drugs had no significant effect on formalin‐evoked nociceptive behaviour in NFC rats (Figure 6A).

Figure 6.

Figure 6

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(A) Effects of fear conditioning and bilateral administration of URB597, AM251 or URB597 + AM251 directly into the PrL on formalin‐evoked nociceptive behaviour in rats over the full 30 min testing period subdivided into 10 min time bins. (B) Effects of fear conditioning and bilateral administration of URB597, AM251 or URB597 + AM251 directly into the PrL on the duration of freezing in formalin‐injected rats over the full 30 min testing period, subdivided into 10 min time bins. (C) Effects of fear conditioning and bilateral administration of URB597, AM251 or URB597 + AM251 directly into the PrL on the distance moved (cm) in formalin‐injected rats over the full 30 min testing period, subdivided into 10 min time bins. All data are expressed as mean ± SEM. *P < 0.05, significantly different from NFC; # P < 0.05, significantly different from FC VEH.

In the ACC study, there was a significant effect of fear conditioning but not treatment or fear conditioning * treatment on CPS over the course of the 30 min testing period (Figure 7A). There was also a significant effect of time and time * fear conditioning but not time * treatment or time * fear conditioning * treatment. Further post hoc analysis with Tukey's test revealed that FC VEH‐treated rats displayed significant less formalin‐evoked nociceptive behaviour compared with NFC VEH‐treated rats in the first 20 min of the testing period, confirming the expression of FCA. Intra‐ACC administration of AM251 or URB597, alone or in combination, had no significant effect on the expression of formalin‐evoked nociceptive behaviour per se or FCA (Figure 7A).

Figure 7.

Figure 7

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(A) Effects of fear conditioning and bilateral administration of URB597, AM251 or URB597 + AM251 directly into the ACC on formalin‐evoked nociceptive behaviour in rats over the full 30 min testing period subdivided into 10 min time bins. (B) Effects of fear conditioning and bilateral administration of URB597, AM251 or URB597 + AM251 directly into the ACC on the duration of freezing in formalin‐injected rats over the full 30 min testing period, subdivided into 10 min time bins. (C) Effects of fear conditioning and bilateral administration of URB597, AM251 or URB597 + AM251 directly into the ACC on the distance moved (cm) in formalin‐injected rats over the full 30 min testing period, subdivided into 10 min time bins. . All data are expressed as mean ± SEM. *P < 0.05, significantly different from NFC.

Data from these sets of experiments are summarised inTable 2.

Table 2.

Summary of the drug effects on the expression of FCA in rats

IfL PrL ACC
VEH − − −
AM251 ↓ ↓ −
URB597 ↓ ↑ −
URB597 + AM251 ↓ (trend) − −
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–, no effect; ↓, attenuated; ↑, enhanced or prolonged.

Effects of intra‐mPFC administration of AM251, URB597 or URB597 + AM251 on the expression of conditioned fear behaviour in the presence of formalin‐evoked nociceptive tone

In the IfL study, there was a significant effect of fear conditioning, treatment and fear conditioning * treatment on the duration of freezing over the course of the 30 min testing period (Figure 5B). There was also a significant effect of time, time * fear conditioning, time * treatment and time * fear conditioning * treatment. Further post hoc analysis using Tukey's test revealed that FC VEH‐treated rats displayed significantly more freezing compared with NFC VEH‐treated rats in the first 20 min of the testing period. Intra‐IfL administration of AM251 or URB597 alone significantly attenuated this contextually induced freezing over the first 20 min of the trial and during 10–20 min when the drugs were co‐administered as shown in Figure 5B.

In the PrL study, there was a significant effect of fear conditioning but not treatment or fear conditioning * treatment on the duration of freezing over the course of the 30 min testing period (Figure 6B). There was also a significant effect of time and time * fear conditioning but not time * treatment or time * fear conditioning * treatment. Further post hoc analysis using Tukey's test revealed that FC VEH‐treated rats displayed significantly more freezing compared with NFC VEH‐treated rats in the first 10 min of the testing period. Intra‐PrL administration of AM251 or URB597, alone or in combination, had no significant effect on the expression of this contextually induced freezing (Figure 6B).

In the ACC study, analysis of the results showed a significant effect of fear conditioning but not treatment or fear conditioning * treatment on the duration of freezing over the course of the 30 min testing period. There was also a significant effect of time and time * fear conditioning but not time * treatment or time * fear conditioning * treatment. Further post hoc analysis using Tukey's test revealed that FC VEH‐treated rats displayed significantly more freezing compared with NFC VEH‐treated rats in the first 10 min of the testing period. Intra‐ACC administration of AM251 or URB597, alone or in combination, had no significant effect on the duration of freezing (Figure 7B).

Data from these sets of experiments are summarised in Table 3.

Table 3.

Summary of the drug effects on the expression of contextually induced freezing in rats

IfL PrL ACC
VEH − − −
AM251 ↓ − −
URB597 ↓ − −
URB597 + AM251 − (0–10 min) − −
↓ (10–20 min)
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–, no effect; ↓, attenuated; ↑, enhanced or prolonged.

Effects of fear conditioning and AM251, URB597 or URB597 + AM251 on locomotor activity in formalin‐treated rats

In the IfL study, there was no significant effect of fear conditioning, treatment or fear conditioning * treatment on distance moved. There was an effect of time and time * fear conditioning but not time * treatment or time * fear conditioning * treatment. Further post hoc analysis revealed no significant differences between‐groupmeans (Figure 5C).

In the PrL study, there was no significant effect of fear conditioning, treatment or fear conditioning * treatment on distance moved. There was an effect of time and time * fear conditioning but not time * treatment or time * fear conditioning * treatment. Further post hoc analysis revealed no significant between‐group differences (Figure 6C).

In the ACC study, there was no significant effect of fear conditioning, treatment or fear conditioning * treatment on distance moved (Figure 7C). There was an effect of time * fear conditioning but not time, time * treatment or time * fear conditioning * treatment. Further post hoc analysis revealed no significant differences between group means (Figure 7C).

Discussion

The data presented here demonstrate for the first time that the endocannabinoid system in the mPFC is an important neural substrate regulating expression of FCA and fear–pain interactions. The results indicate that this regulation occurs in a subregion‐specific manner, with the endocannabinoid system in the PrL, IfL and ACC playing distinct and differential modulatory roles in expression of FCA and fear in the presence of nociceptive tone.

Blockade of CB1 receptors in the PrL with AM251 attenuated FCA with no effects on the expression of formalin‐evoked nociceptive behaviour per se or on contextually induced freezing in the presence of nociceptive tone. In addition, intra‐PrL administration of the FAAH inhibitor URB597 prolonged the expression of FCA in the absence but not the presence of AM251. These findings together suggest that FCA is mediated by endocannabinoids acting at CB1 receptors within the PrL and suggest that the PrL can be considered as an additional important neural substrate for endocannabinoid‐mediated FCA, alongside the ventral hippocampus (Ford et al., 2011), dorsolateral periaqueductal grey (Olango et al., 2012) and basolateral amygdala (Roche et al., 2007; Rea et al., 2013). The results of this PrL study also corroborate our previous studies (Finn et al., 2004; Roche et al., 2007; Roche et al., 2010; Rea et al., 2014b) and those of others (Kinscheck et al., 1984; Helmstetter and Fanselow, 1987), demonstrating that pain‐related behaviour in FC animals can be altered independently of the level of fear being expressed in these animals in the presence of nociceptive tone.

In contrast to the PrL, either CB1 receptor blockade or FAAH inhibition within the IfL attenuated expression of both FCA and contextually induced freezing in the presence of nociceptive tone. Thus, in contrast to the results for the PrL study where the effects of the endocannabinoid system on FCA and fear‐related freezing were dissociable, the endocannabinoid system in the IfL appears to modulate FCA and contextually induced freezing in the same direction. It is also interesting that in the IfL, both AM251 and URB597 attenuated FCA and freezing, given the differing mechanisms of action of these drugs (CB1 receptor blockade vs. FAAH inhibition, respectively). In both the IfL and PrL, AM251 attenuated FCA, suggesting that CB1 receptors in both of these mPFC subregions mediate FCA. While our data provide the first evidence for a role of the endocannabinoid system in the IfL and PrL in mediating conditioned fear‐induced analgesia, previous research has demonstrated a role for the endocannabinoid system in the PrL in the expression of unconditioned fear‐induced analgesia (Freitas et al., 2013). CB1 receptors in the PrL therefore appear to play a key role in mediating the expression of endogenous analgesia to both conditioned and unconditioned aversive stimuli. However, while the data from our PrL study are compatible with the idea that endocannabinoid‐CB1 receptor signalling mediates FCA, the attenuation of FCA following intra‐IfL administration of URB597 is not compatible with the effects of AM251 in the IfL and suggests that activation of a non‐CB1 receptor target by one or more FAAH substrates may instead be mediating the effects of URB597 in the IfL. In this respect, FAAH substrates including AEA, PEA and OEA have been shown to activate a number of non‐CB1 receptors either directly or indirectly (via substrate competition at FAAH) including CB2 receptors (Felder et al., 1996; Griffin et al., 2000; Petrosino and Di Marzo, 2017), TRPV1 channels (Smart et al., 2000; De Petrocellis et al., 2001; Di Marzo et al., 2001; Ross et al., 2001), PPARs (LoVerme et al., 2005; O'Sullivan and Kendall, 2010; Pistis and O'Sullivan, 2017) and GPR55 (Pertwee, 2007; Ryberg et al., 2007; Sharir and Abood, 2010; Kramar et al., 2017). Further studies should address the potential role of these receptors within the IfL in regulation of fear, pain and FCA.

In our ACC study, in contrast to the results obtained for the PrL and IfL, neither AM251 nor URB597 affected the expression of FCA‐ or contextually‐induced freezing, providing further support for the contention that the endocannabinoid system within the mPFC regulates FCA in a subregion‐specific manner. In addition, the data revealed that regardless of the region injected, URB597 and AM251 had no effect on the distance moved, indicating that their effects within the IfL and PrL on FCA‐ and contextually‐induced freezing are likely to represent specific effects on nociceptive and fear‐related behaviour rather than overt effects on locomotor activity.

One implication of our findings is that the endocannabinoid system within each of these mPFC subregions may be an important factor contributing to their differential regulation of fear‐related and pain‐related behaviour, which has previously been described. Alterations in endocannabinoid signalling, coupled with differences in circuitry within, and projections to and from, each of these three mPFC subregions (Vertes, 2002; Vertes, 2004; Hoover and Vertes, 2007), are likely to underlie their different roles in fear, pain and FCA. One of the first reports of specific but differential functions of subregions in the mPFC was by Vidal‐Gonzalez et al. (2006). They found that microstimulation of the PrL enhanced the expression of conditioned fear to a tone and prevented extinction, while microstimulation of the IfL reduced the expression of conditioned fear and microstimulation of the ACC had no effect on either expression or extinction of conditioned fear (Vidal‐Gonzalez et al., 2006). Inactivation of the PrL but not IfL depressed fear responses, while inactivation of the IfL but not PrL impaired the consolidation and retrieval of fear extinction in rats (Laurent and Westbrook, 2009). Lesioning (Kim et al., 2013) or pharmacological inactivation (Corcoran and Quirk, 2007; Sierra‐Mercado et al., 2011) of the PrL impairs the expression of conditioned fear without affecting extinction, while inactivation of the IfL has no effect on fear expression but impairs the acquisition of extinction as well the extinction memory (Sierra‐Mercado et al., 2011). Fewer studies have compared the respective roles of these mPFC regions in modulating pain. Preconditioning and post‐conditioning muscimol‐mediated inactivation of IfL and PrL had no effect on expression of formalin‐evoked pain per se but differentially affected formalin‐evoked condition place aversion, which was impaired by PrL, not IfL, inactivation (Jiang et al., 2014). Similarly, in the present studies, we have observed little or no effect of pharmacological modulation of the endocannabinoid system within these three mPFC subregions on formalin‐evoked nociceptive behaviour in the absence of fear under the conditions of testing used here (but see Okine et al., 2016 where test conditions differed and intra‐ACC administration of AM251 reduced formalin‐evoked nociceptive behaviour). Future studies should address whether this differential modulation of FCA is achieved via differential modulation of the pathways connecting these mPFC subregions to downstream components of the descending inhibitory pain pathway, such as the amygdala and PAG, or more locally within the three subregions themselves via alteration of incoming ascending nociceptive information.

In conclusion, the present studies provide new evidence to support a role for the endocannabinoid system within the subregions of the mPFC in the expression of FCA and conditioned fear in the presence of nociceptive tone. Furthermore, our data suggest that endocannabinoid‐mediated regulation of these behaviours occurs in an mPFC subregion‐specific manner. Elucidation of the role of the endocannabinoid system in different subregions of the mPFC in fear–pain interactions may facilitate increased understanding of, and development of new therapeutic approaches for, pain‐related and fear‐related disorders and their comorbidity.

Author contributions

K.R., F.M., L.C., M.R. and D.P.F. all contributed to the study design and preparation of the manuscript. K.R., F.M. and L. C. collected the data. L.C. analysed the data. M.R. and D P.F. supervised the work and contributed to the data interpretation.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgements

This work was funded by grants from the Science Foundation Ireland (10/IN.1/B2976), the Irish Research Council and a PhD scholarship from the College of Medicine, National University of Ireland Galway.

Rea, K. , McGowan, F. , Corcoran, L. , Roche, M. , and Finn, D. P. (2019) The prefrontal cortical endocannabinoid system modulates fear–pain interactions in a subregion‐specific manner. British Journal of Pharmacology, 176: 1492–1505. 10.1111/bph.14376.

References

  1. Alexander SPH, Christopoulos A, Davenport AP, Kelly E, Marrion NV, Peters JA et al (2017a). The Concise Guide to PHARMACOLOGY 2017/18: G protein‐coupled receptors. Br J Pharmacol 174: S17–S129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander SPH, Fabbro D, Kelly E, Marrion NV, Peters JA, Faccenda E et al (2017b). The Concise Guide to PHARMACOLOGY 2017/18: Enzymes. Br J Pharmacol 174: S272–S359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Almada RC, Coimbra NC, Brandao ML (2015). Medial prefrontal cortex serotonergic and GABAergic mechanisms modulate the expression of contextual fear: intratelencephalic pathways and differential involvement of cortical subregions. Neuroscience 284: 988–997. [DOI] [PubMed] [Google Scholar]
  4. Baulmann J, Spitznagel H, Herdegen T, Unger T, Culman J (2000). Tachykinin receptor inhibition and c‐Fos expression in the rat brain following formalin‐induced pain. Neuroscience 95: 813–820. [DOI] [PubMed] [Google Scholar]
  5. Butler RK, Finn DP (2009). Stress‐induced analgesia. Prog Neurobiol 88: 184–202. [DOI] [PubMed] [Google Scholar]
  6. Butler RK, Nilsson‐Todd L, Cleren C, Lena I, Garcia R, Finn DP (2011). Molecular and electrophysiological changes in the prefrontal cortex–amygdala–dorsal periaqueductal grey pathway during persistent pain state and fear‐conditioned analgesia. Physiol Behav 104: 1075–1081. [DOI] [PubMed] [Google Scholar]
  7. Butler RK, Rea K, Lang Y, Gavin AM, Finn DP (2008). Endocannabinoid‐mediated enhancement of fear‐conditioned analgesia in rats: opioid receptor dependency and molecular correlates. Pain 140: 491–500. [DOI] [PubMed] [Google Scholar]
  8. Connell K, Bolton N, Olsen D, Piomelli D, Hohmann AG (2006). Role of the basolateral nucleus of the amygdala in endocannabinoid‐mediated stress‐induced analgesia. Neurosci Lett 397: 180–184. [DOI] [PubMed] [Google Scholar]
  9. Corcoran KA, Quirk GJ (2007). Activity in prelimbic cortex is necessary for the expression of learned, but not innate, fears. J Neurosci 27: 840–844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Corcoran L, Roche M, Finn DP (2015). The role of the brain's endocannabinoid system in pain and its modulation by stress. Int Rev Neurobiol 125: 203–255. [DOI] [PubMed] [Google Scholar]
  11. Curtis MJ, Alexander S, Cirino G, Docherty JR, George CH, Giembycz MA et al (2018). Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers. Brit J Pharmacol 175: 987–993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. de Novellis V, Palazzo E, Rossi F, De Petrocellis L, Petrosino S, Guida F et al (2008). The analgesic effect of N‐arachidonoyl‐serotonin, a FAAH inhibitor and TRPV1 receptor antagonist, associated with changes in rostral ventromedial medulla and locus coeruleus cell activity in rats. Neuropharmacology 55: 1105–1113. [DOI] [PubMed] [Google Scholar]
  13. De Petrocellis L, Bisogno T, Maccarrone M, Davis JB, Finazzi‐Agro A, Di Marzo V (2001). The activity of anandamide at vanilloid VR1 receptors requires facilitated transport across the cell membrane and is limited by intracellular metabolism. J Biol Chem 276: 12856–12863. [DOI] [PubMed] [Google Scholar]
  14. Devane WA, Dysarz FA 3rd, Johnson MR, Melvin LS, Howlett AC (1988). Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol 34: 605–613. [PubMed] [Google Scholar]
  15. Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G et al (1992). Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258: 1946–1949. [DOI] [PubMed] [Google Scholar]
  16. Di Marzo V, Bisogno T, De Petrocellis L (2001). Anandamide: some like it hot. Trends Pharmacol Sci 22: 346–349. [DOI] [PubMed] [Google Scholar]
  17. Ebrahimzadeh M, Haghparast A (2011). Analgesic effects of cannabinoid receptor agonist WIN55,212‐2 in the nucleus cuneiformis in animal models of acute and inflammatory pain in rats. Brain Res 1420: 19–28. [DOI] [PubMed] [Google Scholar]
  18. Egertova M, Cravatt BF, Elphick MR (2003). Comparative analysis of fatty acid amide hydrolase and cb1 cannabinoid receptor expression in the mouse brain: evidence of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid signaling. Neuroscience 119: 481–496. [DOI] [PubMed] [Google Scholar]
  19. Einarsson EO, Pors J, Nader K (2015). Systems reconsolidation reveals a selective role for the anterior cingulate cortex in generalized contextual fear memory expression. Neuropsychopharmacology 40: 480–487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Felder CC, Nielsen A, Briley EM, Palkovits M, Richardson JM, Riggin RM et al (1996). The role of anandamide and related fatty acid ethanolamides as endogenous ligands for the CB1 and CB2 cannabinoid receptors In: Vanderhoek JY. (ed). Frontiers in Bioactive Lipids. Springer US: Boston, MA. [Google Scholar]
  21. Finn DP, Beckett SR, Richardson D, Kendall DA, Marsden CA, Chapman V (2004). Evidence for differential modulation of conditioned aversion and fear‐conditioned analgesia by CB1 receptors. Eur J Neurosci 20: 848–852. [DOI] [PubMed] [Google Scholar]
  22. Finn DP, Jhaveri MD, Beckett SR, Madjd A, Kendall DA, Marsden CA et al (2006). Behavioral, central monoaminergic and hypothalamo–pituitary–adrenal axis correlates of fear‐conditioned analgesia in rats. Neuroscience 138: 1309–1317. [DOI] [PubMed] [Google Scholar]
  23. Finn DP, Jhaveri MD, Beckett SR, Roe CH, Kendall DA, Marsden CA et al (2003). Effects of direct periaqueductal grey administration of a cannabinoid receptor agonist on nociceptive and aversive responses in rats. Neuropharmacology 45: 594–604. [DOI] [PubMed] [Google Scholar]
  24. Ford GK, Finn DP (2008). Clinical correlates of stress‐induced analgesia: evidence from pharmacological studies. Pain 140: 3–7. [DOI] [PubMed] [Google Scholar]
  25. Ford GK, Kieran S, Dolan K, Harhen B, Finn DP (2011). A role for the ventral hippocampal endocannabinoid system in fear‐conditioned analgesia and fear responding in the presence of nociceptive tone in rats. Pain 152: 2495–2504. [DOI] [PubMed] [Google Scholar]
  26. Freitas RL, Salgado‐Rohner CJ, Hallak JE, Crippa JA, Coimbra NC (2013). Involvement of prelimbic medial prefrontal cortex in panic‐like elaborated defensive behaviour and innate fear‐induced antinociception elicited by GABAA receptor blockade in the dorsomedial and ventromedial hypothalamic nuclei: role of the endocannabinoid CB1 receptor. Int J Neuropsychopharmacol 16: 1781–1798. [DOI] [PubMed] [Google Scholar]
  27. Fuchs PN, Peng YB, Boyette‐Davis JA, Uhelski ML (2014). The anterior cingulate cortex and pain processing. Front Integr Neurosci 8: 35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gilmartin MR, Balderston NL, Helmstetter FJ (2014). Prefrontal cortical regulation of fear learning. Trends Neurosci 37: 455–464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Gilmartin MR, Kwapis JL, Helmstetter FJ (2013). NR2A‐ and NR2B‐containing NMDA receptors in the prelimbic medial prefrontal cortex differentially mediate trace, delay, and contextual fear conditioning. Learn Mem 20: 290–294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Griffin G, Tao Q, Abood ME (2000). Cloning and pharmacological characterization of the rat CB2 cannabinoid receptor. J Pharmacol Exp Ther 292: 886–894. [PubMed] [Google Scholar]
  31. Guindon J, Hohmann AG (2009). The endocannabinoid system and pain. CNS Neurol Disord Drug Targets 8: 403–421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Harding SD, Sharman JL, Faccenda E, Southan C, Pawson AJ, Ireland S et al (2018). The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY. Nucl Acids Res 46: D1091–D1106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Helmstetter FJ, Fanselow MS (1987). Effects of naltrexone on learning and performance of conditional fear‐induced freezing and opioid analgesia. Physiol Behav 39: 501–505. [DOI] [PubMed] [Google Scholar]
  34. Herkenham M, Lynn AB, Johnson MR, Melvin LS, De Costa BR, Rice KC (1991). Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J Neurosci 11: 563–583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hohmann AG, Suplita RL, Bolton NM, Neely MH, Fegley D, Mangieri R et al (2005). An endocannabinoid mechanism for stress‐induced analgesia. Nature 435: 1108–1112. [DOI] [PubMed] [Google Scholar]
  36. Hoover WB, Vertes RP (2007). Anatomical analysis of afferent projections to the medial prefrontal cortex in the rat. Brain Struct Funct 212: 149–179. [DOI] [PubMed] [Google Scholar]
  37. Jiang ZC, Pan Q, Zheng C, Deng XF, Wang JY, Luo F (2014). Inactivation of the prelimbic rather than infralimbic cortex impairs acquisition and expression of formalin‐induced conditioned place avoidance. Neurosci Lett 569: 89–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Johansen JP, Fields HL, Manning BH (2001). The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci 98: 8077–8082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010). Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kim EJ, Kim N, Kim HT, Choi JS (2013). The prelimbic cortex is critical for context‐dependent fear expression. Front Behav Neurosci 7: 73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kinscheck IB, Watkins LR, Mayer DJ (1984). Fear is not critical to classically conditioned analgesia: the effects of periaqueductal gray lesions and administration of chlordiazepoxide. Brain Res 298: 33–44. [DOI] [PubMed] [Google Scholar]
  42. Kramar C, Loureiro M, Renard J, Laviolette SR (2017). Palmitoylethanolamide modulates GPR55 receptor signaling in the ventral hippocampus to regulate mesolimbic dopamine activity, social interaction, and memory processing. Cannabis Cannabinoid Res 2: 8–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Laurent V, Westbrook RF (2009). Inactivation of the infralimbic but not the prelimbic cortex impairs consolidation and retrieval of fear extinction. Learn Mem 16: 520–529. [DOI] [PubMed] [Google Scholar]
  44. Laviolette SR, Grace AA (2006). The roles of cannabinoid and dopamine receptor systems in neural emotional learning circuits: implications for schizophrenia and addiction. Cell Mol Life Sci 63: 1597–1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Laviolette SR, Lipski WJ, Grace AA (2005). A subpopulation of neurons in the medial prefrontal cortex encodes emotional learning with burst and frequency codes through a dopamine D4 receptor‐dependent basolateral amygdala input. J Neurosci 25: 6066–6075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Lisboa SF, Reis DG, Da Silva AL, Correa FM, Guimaraes FS, Resstel LB (2010). Cannabinoid CB1 receptors in the medial prefrontal cortex modulate the expression of contextual fear conditioning. Int J Neuropsychopharmacol 13: 1163–1173. [DOI] [PubMed] [Google Scholar]
  47. LoVerme J, La Rana G, Russo R, Calignano A, Piomelli D (2005). The search for the palmitoylethanolamide receptor. Life Sci 77: 1685–1698. [DOI] [PubMed] [Google Scholar]
  48. Luongo L, De Novellis V, Gatta L, Palazzo E, Vita D, Guida F et al (2013). Role of metabotropic glutamate receptor 1 in the basolateral amygdala‐driven prefrontal cortical deactivation in inflammatory pain in the rat. Neuropharmacology 66: 317–329. [DOI] [PubMed] [Google Scholar]
  49. Mailleux P, Vanderhaeghen JJ (1992). Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry. Neuroscience 48: 655–668. [DOI] [PubMed] [Google Scholar]
  50. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI (1990). Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346: 561–564. [DOI] [PubMed] [Google Scholar]
  51. McGrath JC, Lilley E (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol 172: 3189–3193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Mechoulam R, Ben‐Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz AR et al (1995). Identification of an endogenous 2‐monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol 50: 83–90. [DOI] [PubMed] [Google Scholar]
  53. Munro S, Thomas KL, Abu‐Shaar M (1993). Molecular characterization of a peripheral receptor for cannabinoids. Nature 365: 61–65. [DOI] [PubMed] [Google Scholar]
  54. O'Sullivan SE, Kendall DA (2010). Cannabinoid activation of peroxisome proliferator‐activated receptors: potential for modulation of inflammatory disease. Immunobiology 215: 611–616. [DOI] [PubMed] [Google Scholar]
  55. Okine BN, Madasu MK, Mcgowan F, Prendergast C, Gaspar JC, Harhen B et al (2016). N‐Palmitoylethanolamide in the anterior cingulate cortex attenuates inflammatory pain behaviour indirectly via a CB1 receptor‐mediated mechanism. Pain 157: 2687–2696. [DOI] [PubMed] [Google Scholar]
  56. Olango WM, Geranton SM, Roche M, Hunt SP, Finn DP (2014). Novel molecular correlates of endocannabinoid‐mediated fear‐conditioned analgesia in rats. Eur J Pain 18: 182–191. [DOI] [PubMed] [Google Scholar]
  57. Olango WM, Roche M, Ford GK, Harhen B, Finn DP (2012). The endocannabinoid system in the rat dorsolateral periaqueductal grey mediates fear‐conditioned analgesia and controls fear expression in the presence of nociceptive tone. Br J Pharmacol 165: 2549–2560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Paxinos G, Watson C (1998). The Rat Brain in Stereotaxic Coordinates. Acadmeic Press: New York. [Google Scholar]
  59. Pertwee RG (2007). GPR55: a new member of the cannabinoid receptor clan? Br J Pharmacol 152: 984–986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Petrosino S, Di Marzo V (2017). The pharmacology of palmitoylethanolamide and first data on the therapeutic efficacy of some of its new formulations. Br J Pharmacol 174: 1349–1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Pistis M, O'Sullivan SE (2017). The role of nuclear hormone receptors in cannabinoid function. Adv Pharmacol 80: 291–328. [DOI] [PubMed] [Google Scholar]
  62. Rea K, Ford GK, Olango WM, Harhen B, Roche M, Finn DP (2014a). Microinjection of 2‐arachidonoyl glycerol into the rat ventral hippocampus differentially modulates contextually induced fear, depending on a persistent pain state. Eur J Neurosci 39: 435–443. [DOI] [PubMed] [Google Scholar]
  63. Rea K, Lang Y, Finn DP (2009). Alterations in extracellular levels of gamma‐aminobutyric acid in the rat basolateral amygdala and periaqueductal gray during conditioned fear, persistent pain and fear‐conditioned analgesia. J Pain 10: 1088–1098. [DOI] [PubMed] [Google Scholar]
  64. Rea K, Olango WM, Harhen B, Kerr DM, Galligan R, Fitzgerald S et al (2013). Evidence for a role of GABAergic and glutamatergic signalling in the basolateral amygdala in endocannabinoid‐mediated fear‐conditioned analgesia in rats. Pain 154: 576–585. [DOI] [PubMed] [Google Scholar]
  65. Rea K, Olango WM, Okine BN, Madasu MK, Mcguire IC, Coyle K et al (2014b). Impaired endocannabinoid signalling in the rostral ventromedial medulla underpins genotype‐dependent hyper‐responsivity to noxious stimuli. Pain 155: 69–79. [DOI] [PubMed] [Google Scholar]
  66. Rea K, Roche M, Finn DP (2011). Modulation of conditioned fear, fear‐conditioned analgesia, and brain regional c‐Fos expression following administration of muscimol into the rat basolateral amygdala. J Pain 12: 712–721. [DOI] [PubMed] [Google Scholar]
  67. Resstel LB, Joca SR, Guimaraes FG, Correa FM (2006). Involvement of medial prefrontal cortex neurons in behavioral and cardiovascular responses to contextual fear conditioning. Neuroscience 143: 377–385. [DOI] [PubMed] [Google Scholar]
  68. Roche M, Johnston P, Mhuircheartaigh ON, Olango WM, Mackie K, Finn DP (2010). Effects of intra‐basolateral amygdala administration of rimonabant on nociceptive behaviour and neuronal activity in the presence or absence of contextual fear. Eur J Pain 14: 487–495. [DOI] [PubMed] [Google Scholar]
  69. Roche M, O'Connor E, Diskin C, Finn DP (2007). The effect of CB1 receptor antagonism in the right basolateral amygdala on conditioned fear and associated analgesia in rats. Eur J Neurosci 26: 2643–2653. [DOI] [PubMed] [Google Scholar]
  70. Ross RA, Gibson TM, Brockie HC, Leslie M, Pashmi G, Craib SJ et al (2001). Structure–activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens. Br J Pharmacol 132: 631–640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Ryberg E, Larsson N, Sjogren S, Hjorth S, Hermansson NO, Leonova J et al (2007). The orphan receptor GPR55 is a novel cannabinoid receptor. Br J Pharmacol 152: 1092–1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Sharir H, Abood ME (2010). Pharmacological characterization of GPR55, a putative cannabinoid receptor. Pharmacol Ther 126: 301–313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Sharpe M, Killcross S (2014). The prelimbic cortex uses contextual cues to modulate responding towards predictive stimuli during fear renewal. Neurobiol Learn Mem 118C: 20–29. [DOI] [PubMed] [Google Scholar]
  74. Sierra‐Mercado D, Padilla‐Coreano N, Quirk GJ (2011). Dissociable roles of prelimbic and infralimbic cortices, ventral hippocampus, and basolateral amygdala in the expression and extinction of conditioned fear. Neuropsychopharmacology 36: 529–538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI et al (2000). The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol 129: 227–230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K et al (1995). 2‐Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun 215: 89–97. [DOI] [PubMed] [Google Scholar]
  77. Suplita RL 2nd, Farthing JN, Gutierrez T, Hohmann AG (2005). Inhibition of fatty‐acid amide hydrolase enhances cannabinoid stress‐induced analgesia: sites of action in the dorsolateral periaqueductal gray and rostral ventromedial medulla. Neuropharmacology 49: 1201–1209. [DOI] [PubMed] [Google Scholar]
  78. Tsou K, Brown S, Sanudo‐Pena MC, Mackie K, Walker JM (1998). Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience 83: 393–411. [DOI] [PubMed] [Google Scholar]
  79. Vertes RP (2002). Analysis of projections from the medial prefrontal cortex to the thalamus in the rat, with emphasis on nucleus reuniens. J Comp Neurol 442: 163–187. [DOI] [PubMed] [Google Scholar]
  80. Vertes RP (2004). Differential projections of the infralimbic and prelimbic cortex in the rat. Synapse 51: 32–58. [DOI] [PubMed] [Google Scholar]
  81. Vidal‐Gonzalez I, Vidal‐Gonzalez B, Rauch SL, Quirk GJ (2006). Microstimulation reveals opposing influences of prelimbic and infralimbic cortex on the expression of conditioned fear. Learn Mem 13: 728–733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Wang HC, Wang YC, Huang AC, Chai SC, Wu YS, Wang CC (2009). Roles of corticosterone in formalin‐induced conditioned place aversion in rats. Neurosci Lett 464: 122–126. [DOI] [PubMed] [Google Scholar]
  83. Watson GS, Sufka KJ, Coderre TJ (1997). Optimal scoring strategies and weights for the formalin test in rats. Pain 70: 53–58. [DOI] [PubMed] [Google Scholar]

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