The Contribution of Baseline Circulating Endocannabinoids to Individual Differences in Human Pain Sensitivity: A Quantitative Sensory Testing Study

The endocannabinoid (eCB) system —comprising cannabinoid receptors, eCBs (anandamide — AEA, 2 -arachidonoylglycerol—2-AG) and related N-acylethanolamines (NAEs; N- palmitoylethanolamide—PEA, and N-oleoylethanolamide—OEA), and metabolizing enzymes (e.g., fatty acid amide hydrolase; FAAH)—modulates nociceptive circuits in rodents. In humans, the FAAH C385A polymorphism is associated with reduced pain sensitivity, suggesting eCB tone influences individual pain differences, but this has yet to be tested. Here, we de termined whether the eCB system is associated with somatosensory and pain sensitivity measured with quantitative sensory testing (QST) in 91 healthy participants (39 males, 52 females). We tested three hypotheses: (1) FAAH C385A polymorphism, cannabis use, and sex affect serum eCB/NAE concentrations; (2) FAAH C385A carriers show altered pain sensitivity versus non -carriers; and (3) baseline serum eCB/NAE concentrations are associated with QST measures. eCB/NAE concentrations were not statistically different based on sex (p > .05), based on FAAH genotype (p > .05), and based on cannabis use ( p > .05). To address collinearity of AEA, OEA and PEA in linear regression analyses, we performed a factor analysis with principal components analysis, which identified a single component of FAAH substrates. Linear regressions found that FAAH genotype did not affect QST measures and that baseline 2-AG and FAAH substrate concentrations were not associated with QST measures, except pressure pain thresholds (PPT; p = 0.003), which were associated with AEA and OEA. Baseline eCB/NAE levels may not be a global predictor of QST somatosensory and pain tests in healthy adult humans ; nonetheless, circulating FAAH substrate levels were associated with PPT. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 3

Pain, Quantitative Sensory Testing, N-acylethanolamines, Fatty-acid amide hydrolase, Sex Differences .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 4

There are significant individual differences in human pain sensitivity [16,19,32,63]. Biological factors such as sex, the state of the nociceptive and pain modulatory systems, and genetic variability contribute to individual differences in pain [4,17,19,20,26,51]. Neuromodulators that affect nociceptive pathways and descending modulatory circuits may thus contribute to individual differences in pain sensitivity. One such neuromodulatory system identified in preclinical models of pain is the endocannabinoid (eCB) system [21,28,39,81,90]. The eCB system includes cannabinoid receptors; endogenous cannabinoid ligands, including 2-arachidonoylglycerol (2-AG), N-arachidonoylethanolamine/anandamide (AEA), and related N-acylethanolamines (NAEs) including N-palmitoylethanolamide (PEA) and N- oleoylethanolamide (OEA); and metabolizing enzymes, including fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase. Although NAEs are not strictly eCBs, they may also have pain modulatory effects , both directly through peroxisome proliferator-activated receptors [72], and indirectly via elevation of AEA levels through substrate competition at FAAH [3,45,52,65]. Preclinical research has shown that eCBs and NAEs are present at every level of the nociceptive system , including within key structures of descending modulatory circuits [10,37,66,67,81,90]. In rodents, eCBs modulate rodent thermal and mechanical sensitivity [23,55,56]. Further, there are sex differences in eCB ligand concentrations in the nociceptive and modulatory pathways [53] and effects on pain [8,86]. However, the contribution of eCBs to pain sensitivity in healthy humans remains unknown. Evidence supporting the role of the eCB system in human pain comes from studies of the functional FAAH rs324420 (C385A) polymorphism, which can result in elevated AEA, OEA and PEA levels [58,79]. Variants of the FAAH gene are associated with lower cold pain sensitivity , reduced postoperative analgesic needs compared to non-carriers [14], and congenital pain insensitivity [93]. Further, CB1 receptor antagonism selectively reduced non-opioidergic placebo analgesia [7,70]. Thus, natural eCB system variation may account for individual pain sensitivity differences. However, circulating eCB concentrations are affected by cannabis use [44], and may thus modify the relationship with pain sensitivity, but this remains unknown. Individual differences in pain sensitivity can be measured with quantitative sensory testing (QST) [47,74- 76]—a standardized set of psychophysical tests to assess multiple sensory modalities (innocuous and noxious thermal and mechanical stimuli). Here, we explore whether the eCB system is associated with individual QST measures. We first explore whether the FAAH C385A polymorphism, sex, and cannabis use affect circulating eCB/NAE levels. Based on the literature, we expect that those with the C385A SNP have higher levels of FAAH substrates (AEA, OEA, and PEA) [18,78,80], and that females have lower levels of FAAH substrates than males [2,53]. Next, we test whether individuals with the FAAH C385A polymorphism show different pain sensitivity than individuals without the polymorphism. We hypothesize that A-allele carriers have lower pain sensitivity. Third, we test relationships between baseline circulating eCB/NAE levels and QST measures, hypothesizing that higher eCB/NAE concentrations correlate with lower pain sensitivity. Finally, we explore whether there are sex differences in these relationships, anticipating that there will be differences, but without directional predictions. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 5

Participants One hundred and ten healthy adults (62 females and 48 males) aged between 21 and 45 years of age were recruited from the University of Toronto and surrounding community. All participants consented to procedures approved by the University of Toronto’s Human Research Ethics Board (Protocol #38322). The sample size we aimed to recruit was 100 participants, as this was the first study of its kind. Participants were screened against the following exclusion criteria with self-report: current pain, history of chronic pain, chronic illness, psychiatric disorder or neurological disorder s, pregnancy, breastfeeding, immunocompromised, specific dietary restrictions (that would not allow participants to eat the carbohydrate snack provided, see below), < 20 years of age, and ≥ 45 years of age (to mitigate any effects of cutaneous sensory changes that occur with aging, and can affect pain sensitivity). Participants were asked to refrain from any cannabis -use 24 hours prior to the session, to minimize the effects of Δ9-tetrahydrocannabinol (Δ9-THC) on pain. Given the high prevalence of recreational cannabis use in Canada following legalization [41], complete exclusion of cannabis users was deemed impractical. Additionally, as dietary fats lead to the synthesis of some eCBs and fatty acid amides, participants were asked to fast overnight (for 12 hours) prior to the study session. To control for the dynamic levels of circulating eCBs and NAEs that exhibit a circadian rhythm [42], all the sessions took place between 9:00AM and 12:00PM. Experimental Session The data presented in this study are part of a larger study with an MRI scanning session. The MRI data are outside the scope of this study. We describe the entire experimental session for completeness. Upon arrival to the Centre for Multimodal Sensorimotor and Pain Research, University of Toronto, and after consent, fasted participants were given the choice of a 30g blueberry oat bar or 30g chocolate chip oat bar (MadeGood© Mornings, Riverside Natural Fo ods, Toronto, ON, Canada) to control for the nutritional value of the snack. Participants then completed two additional screening questionnaires: the Beck Depression Inventory II (BDI -II) and the Mini Mental State Exam (MMSE). Participants then completed a battery of questionnaires (see Questionnaires below), provided a blood sample, and finally underwent a QST battery. Figure 1 provides a summary of the experimental session. A subset of 99 participants were then taken to the MRI facility by taxi and underwent a brain imaging session (outside the scope of this study). Participants were financially compensated for their participation in the study. Figure 1: Schematic representation of the experimental session. Note that the offset analgesia and conditioned pain modulation are not included herein as they are the focus of another study. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 6 Screening Questionnaires The BDI-II is a 21 -item self-report questionnaire that allows individuals to rate their agreement with statements about thoughts and feelings in daily life and is used to measure attitudes and symptoms of depression [6]. Participants who scored > 20 on the questionnaire, demonstrating moderate depression were briefly consulted to ensure that they are feeling well enough to proceed with the experiment, and if they agreed to continue, they were included in the study. Those who indicated suicidal ideation (score > 1 on item 9) were excluded. We also collected the Patient Health Questionnaire (PHQ-9), a multipurpose instrument that allows screening of depression and serves as a tool to diagnose and monitor the severity of dep ression [50]. This test supplemented the BDI to screen participants for depression, and to align protocols with other studies to increase the utility of the data when published for open access use. The MMSE is a tool for systematically and thoroughly assessing mental status. The MMSE is an 11 -item questionnaire that can be used to ensure there are no cognitive impairments by measuring five areas of cognitive function: registration, orientation, recall, attent ion and calculation, and language [33]. This questionnaire served to ensure that participants were alert and could follow directives prior to beginning the experiment. Questionnaires and Measures We collected several questionnaires to capture factors associated with pain sensitivity or circulating eCB/NAE concentrations. Related to the current study, participants completed a demographics questionnaire, and the self -report Daily Sessions, Frequency, Age of Onset, and Quantity of Cannabis Use Inventory (DFAQ-CU) [25]. The demographics questionnaire captured participants’ a ge, sex, gender, and highest level of education attained. The DFAQ -CU is a psychometrically sound inventory which assesses the frequency, age of onset, and quantity of cannabis use. In this study the DFAQ-CU was used to assess participants’ history of cannabis use. As the scale does not provide a continuous outcome measure, we summarized each participant’s score on an ordinal scale: 0 = never used cannabis; 1 = previously used cannabis, but not in the last 3 months; 2 = currently using cannabis. Participants also had their height and weight measured to calculate body mass index (BMI). Participants also completed several other questionnaires outside the scope of this study, which we list here for completeness: the Pain Catastrophizing Scale, Fear of Pain Questionnaire– III, Positive and Negative Affect Scale, Action Control Scale, the PROMIS Anxiety Short Form 7a, and the State-Trait Anxiety Inventory. Blood Sample Collection and Processing Blood samples were collected from the participants by certified phlebotomy technicians. Two vials of blood were collected: 2 mL sample into BD Vacutainer® spray coated K2EDTA 3.6 mg blood tubes, and a 6 mL sample in a BD Vacutainer® Serum tube. The samples were gently inverted 10 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 7 times following collection. The 2 mL sample was immediately snap frozen on dry ice and kept at - 80˚C until shipment. The 6mL sample was left to clot at room temperature for 30 minutes. Next, this sample was centrifuged at 1400 g for 15 minutes at 4˚C. The serum layer was collected with a disposable transfer pipette and aliquoted into 2 labelled 1.5 mL microcentrifuge tubes. The samples were snap-frozen on dry ice and kept at -80˚C until analysis. Samples were shipped in dry ice to the University of Galway, Ireland for eCB/NAE quantification and FAAH C385A genotyping. Circulating Endocannabinoid Quantification Serum eCBs and NAEs were quantified using liquid chromatography -tandem mass spectrometry (LC-MS/MS) as described previously, with some modifications [5,11]. Briefly, 20 μL 100% acetonitrile containing deuterated internal standards (50 ng of 2-AG-d8, 2.5 ng of AEA-d8, 2.5 ng of OEA-d4 and 2.5 ng of PEA -d4) were added to each sample. Samples were then vortexed and allowed to equilibrate for 10 minutes on ice. For protein precipitation, 1 ml of 100% ACN containing 0.1% formic acid, maintained at 4°C, was added to each sample and immediately vortexed and incubated on ice for 30 minutes. The precipitated proteins were pelleted by centrifugation at 18,620 g for 15 minutes at 4°C. A filter (FisherbrandTM Non-sterile PTFE Hydrophyl, 25 mm, 0.45 µM Syringe Filter, Fisher Scientific, Ireland) was placed in a 5 ml SafeSeal tube (SARSTEDT, Ireland) for each sample. 1 ml syringes were attached to the filters by carefully inserting the syringe neck into the filter inlet. The plunger/piston from each 1 mL syringe was removed and saved in a sterile container. 1100 µL of the supernatant was removed without disturbing the pellet and loaded into the syringe. The supernatant was allowed to flow through the filter until the syringe was emptied. Once the supernatant had flown through the syringe into the microcentrifuge tube, 700 µL of 100% ACN was added to the syringe to displace the dead volume of the filter. The syringe plunger was slowly re-inserted, and the 100% ACN was pushed through the filter. The collected eluate was vortexed, and 500 µL of the total volume was transferred to a new 1.5 mL microcentrifuge tube and dried down at 45°C for approximately 1 hour in a centrifugal concentrator (Eppendorf Concentrator plus complete system, Davidson and Hardy Ltd, Ireland). Each sample was reconstituted in 20 µL of 100% ACN before transferring it to HPLC vials and were then separated on a Zorbax ® C18 column (150 × 0.5 mm internal diameter; Agilent Technologies, Cork, Ireland) by reversed -phase gradient elution initially with a mobile phase of 65% acetonitrile and 0.1% formic acid, which was ramped linearly up to 100% acetonitrile and 0.1% formic acid over 10 min. Under these conditions, 2 -AG, AEA, PEA and OEA eluted at the following retention times: 6.6 min, 6.9 min, 7.0 min, and 7.2 min, respectively. Analyte detection was carried out in electrospray -positive ionization mode on an Agilent 1260 In finity II HPLC system coupled to a SCIEX QTRAP 4500 mass spectrometer operated in triple quadrupole mode (SCIEX Ltd, Phoenix House Lakeside Drive Centre Park, United Kingdom). was performed by ratiometric analysis and expressed as nmol or pmol per mL of serum. FAAH C385A Genotyping Genomic DNA (gDNA) were extracted using standard protocols (Nucleospin® DNA isolation kit, Macherey-Nagel, Düren, Germany ). All samples were genotyped for the C385A FAAH SNP (rs324420) using predesigned TaqMan primers and universal genotyping master mix (Life Technologies). Genotyping assays were performed according to manufacturers’ protocols using an Applied Biosystems (ABI) StepOne Plus PCR machine and ABI allelic discrimination software. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 8 Quantitative Sensory Testing Thermal QST measures Innocuous Thermal Detection and Pain Thresholds: We measured the participant’s ability to detect thermal stimuli in the innocuous and noxious ranges. We used the T11 thermode with a stimulation area of 9 cm 2 to generate thermal sensations in the cool, warm, noxious cold and noxious heat ranges on the volar forearm with the Thermal Cutaneous Stimulator device (TCS II, QSTLab, Strasbourg, France) [34,91,92]. The thermode was placed on the right volar forearm and delivered thermal stimuli on the participant’s skin to evaluate cool detection threshold (CDT), warm detection threshold (WDT), cold pain threshold (CPT) and heat pain threshold (HPT). For each modality, the thermode started at a baseline temperature of 30°C and increased or decreased at a rate of 1°C/s for CDT, WDT, CPT and HPT protocols [29]. Participants were asked to press a response button when a change in the perception towards the target stimulus was detected for the first time. For innocuous stimuli, we obtained four measurements for each detection threshold, with an interstimulus inter val of 2 seconds. The first trial was discarded in all participants. For pain detection thresholds, we provided participants with the International Association for the Study of Pain’s definition of pain: “an unpleasant sensory and emotional experience asso ciated with, or resembling that associated with, actual or potential tissue damage” [73]. We obtained three measurements for each thermal pain threshold, and all three were included in the analysis. The arithmetic mean was calculated for each thermal detection threshold (in °C). For safety and technical reasons, cold pain thresholds were considered maximal at 0 ˚C, and heat pain thresholds were considered maximal at 50 ˚C. Heat Pain Tolerance (HPTol): Participants underwent a HPTol paradigm with the thermode placed on the right volar forearm of the participant. The stimulus increased at a rate of 1°C/s. Participants were instructed to press the response button whenever they could no longer tolerate the heat pain elicited by the thermal probe. The TCS device was programmed to have an upper limit of 50˚C for safety. Note, that as HPTol was added to the battery of tests after the study had started, n = 11 did not undergo this test and the sample size for the HPTol test was n = 88. Cold Pain Tolerance ( CPTol): CPTol was measured with the cold pressor task. An 18 L water- bath (FBATH18, Techne, Burlington, NJ, USA) with a digital immersion circulator (FTE10DPC, Techne) and a low-temperature cold-pressor with a rigid coil probe (FRU2P, Techne). The water bath was set to 8 ± 1°C. Participants immersed their dominant hand into the bath up until their wrist with their palm facing down and fingers spread out. A timer was set at time of immersion. Participants were instructed to keep their hand in the water as long as possible, up to the point they could no longer tolerate the cold pain. They were instructed to verbally announce “pain” when the sensation of the cold water became painful, and this time was recorded as a cold pain threshold. Once participants removed their hand, they provided a pain intensity rating on a verbal numeric rating scale, rating from 0 (no pain) to 100 (worst pain imaginable). The duration of immersion was set to a maximum time of 180 seconds if participants could fully tolerate the stimulus. CPTol was calculated by subtracting the time of pain threshold from the time at which pain tolerance was maximized. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 9 Mechanical QST measures Pressure Pain Threshold: PPT was assessed using a handheld digital algometer (Algomed ) fitted with a 1 cm² probe. With participants seated upright, the probe was applied manually to three adjacent sites on the dominant upper trapezius. Participants held a response button in their non - dominant hand and were instructed to press it and say “p ain” as soon as the sensation changed from pressure to pain. Each of the three trials was separated by approximately 10 seconds, and the PPT value was defined as the arithmetic mean of these three measurements. Temporal Summation of Pain (TSP) : TSP was conducted on the dominant volar forearm. Three distinct locations no further than 2 cm apart were marked in a triangular shape. Participants were instructed to close their eyes. A single weighted pinprick stimulator of 256 mN with a 0.25 mm diameter contact area (MRC Systems GmbH-Medizintechnische Systeme, Heidelberg, Germany) was used to perform a single pinprick stimulus. Participants reported their perceived pain intensity on a verbal numerical rating scale between 0 (no pain) to 100 (worst pain imaginable). Next, a train of ten 256 mN pinprick stimulations was applied at 1 Hz to the two other marked locations. Following the training of ten repeated stimuli, participants rated their perceived pain from the last stimulus. A 2-minute break was then taken prior to stimulation of the next site. This procedure was repeated a total of three times. The final value for TSP was calculated by taking the difference between each of the ratings after the tenth stimulus and the ratings after the first stimulus and then computed an arithmetic average of those differences. Statistical Analyses All statistical analyses were performed in JASP v.0.19.3[83], unless otherwise noted. Differences in eCB/NAE ligands based on sex, FAAH C385A SNP genotype, and cannabis-use To determine whether there are sex differences in levels of circulating eCBs (AEA, 2 -AG) and NAEs (OEA, PEA) we first tested the residuals for normality with Shapiro -Wilk’s test, with significance set at p < 0.05. Normally distributed variables were compared between sexes with an independent samples t-test. Non -normally distributed variables were compared with a Mann - Whitney U test. Significance was set at p < 0.05. Similarly, to test whether the FAAH C385A SNP had a significant effect on levels of circulating eCBs/NAEs, all eCB/NAE concentrations were first standardized (z‑scored) across participants. We then tested the residuals for normality with Shapiro‑Wilk’s test, with significance set at p < 0.05. Normally distributed variables were compared between those without vs. those with an A allele with an independent samples t-test. Non -normally distributed variables were compared with a Mann-Whitney U test. Significance was set at p < 0.05. Next, we sought to determine whether cannabis-use was associated with baseline levels of circulating eCBs/NAEs. We performed a multivariate analysis of variance (MANOVA) with Pillai’s test, to determine whether z -scored AEA, OEA, PEA and 2 -AG were different between those who have never used cannabis, those who have previously used cannabis, but do not .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 10 currently use, and those who currently use cannabis , based on the DFAQ -CU. Significance was set to p < 0.05. Data Reduction As all three FAAH substrates were highly correlated, and to reduce the number of multiple comparisons, we performed a principal components analysis (PCA). First, the variables were z - transformed. We ensured that criteria for PCA were met with a Kaiser -Meyer-Olkin Test for Sampling Adequacy (> 0.5) and Bartlett’s test of sphericity (p 1 were accepted as a solution. If more than 1 component was identified, these were orthogonalized with a varimax rotation. Linear Regressions All dependent variables (QST test outcome measures) were z-transformed. To determine whether pain sensitivity is associated with levels of eCBs/NAEs , we performed t wo sets of linear regressions: (1) thermal stimuli (WDT, CDT, HPT, CPT , HPTol, CPTol ) and (2) mechanical stimuli (PPT, TSP). Each linear regression had a different dependent variable (the QST measure), and the same set of predictors. Predictors were tested across five additive models. The first (null) model included age and BMI. BMI was included as there is some evidence suggesting associations with eCBs and NAEs [57]. In the second model, sex was added. The third model incorporated the FAAH SNP genotype in the model. Note that as the AA genotype only had 5 participants, we coded the genotype as a binary variable: without (CC) and with (AA and AC) the SNP. The fourth model added the circulating FAAH substrate level composite score and circulating 2-AG levels. The fifth model added cannabis use to the model. This design allows us to test all three hypotheses in a single analysis. Model selection was based on whether the model fit was significant and based on the lowest Aikaike Information Criterion (AIC). All models were checked for collinearity using the variance inflation factor, ensuring it is below a conservative threshold of 2.5. Significance was set to p < 0.05, Bonferroni corrected for all other linear regressions in the test set (i.e., p < 0.0083 for thermal stimuli; p < 0.025 for mechanical stimuli). In cases were the FAAH substrate composite score was associated with a QST measure, post-hoc partial correlation analyses were performed to determine which FAAH substrate s are associated with PPTs, while controlling for age, BMI, sex, FAAH genotype, and 2 -AG levels, to best mimic the linear regression analysis. Significance was set at p < 0.0083.

Participants We recruited 110 participants for this study. Of these , six were excluded due to inability to draw blood, three due to incidental findings in MRI, and two withdrew from the study, resulting in a cohort of 99 participants (44 males and 55 females). Of these, serum eCB/NAEs data were missing for 5 participants (3 males and 2 females), FAAH SNP genotype was not captured in 3 participants (2 males and 1 female) due to technical issues, and thermal detection thresholds were not recorded for 2 males. Furthermore, as the HPTol test was added after the start of the study, these data are missing for 11 participants (7 males and 4 females). Our final sample included 81 participants for .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 11 HPTol, 89 participants for WDT and CDT, and 91 participants for additional analyses (see Figure 2). Figure 2. Flow diagram of recruitment and sample. Eighteen participants had partial datasets. *indicates sample overlap. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 12 Table 1. Demographic and anthropometric characteristics of the study sample. Characteristic Total Sample (N = 91) Age (years), Mean ± SD 25.2 ± 4.9 Sex, n (%) Female 52 (57.1%) Male 39 (42.9%) Gender, n (%) Woman 49 (53.8%) Man 40 (44.0%) Other 2 (2.2%) BMI, Mean ± SD 23.2 ± 3.0 Education Level, n (%) High School or less 14 (15.4%) Some college/Incomplete Bachelor’s 7 (7.7%) Bachelor’s completed 49 (53.8%) Graduate/professional Degree 21 (23.1%) FAAH rs324420 Genotype CC 59 (64.8%) CA 27 (29.7%) AA 5 (5.5%) Cannabis Use, n (%) Has never used 43 (47.3%) Has used in the past 23 (25.2%) Currently using 25 (27.5%) No Effect of Sex, FAAH SNP Genotype, and Cannabis -use on Baseline Circulating eCBs/NAEs We found no significant sex differences in baseline levels of circulating eCBs/NAEs (all p > 0.12); Table S1). Similarly, there were no differences in baseline levels of circulating eCBs/NAEs based on FAAH genotype (all p > 0.06; Table S2). Lastly, a MANOVA showed no significant differences in baseline levels of circulating eCBs/NAEs with cannabis -use comparing non -users, previous users, and current users (F8,172 = 1.60, Pillai’s trace = 0.14, p = 0.13). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 13 Association between QST measures and eCBs/NAEs Data Reduction There was a single resultant component (weightings provided in Table S 3), which we refer to as “FAAH substrates” herein. Thermal QST Measures Distributions of thermal QST measures are presented in Figure 3. Figure 3. Distribution of Thermal QST measures across participants. Each bar represents a single participant, and participants are ranked in ascending order. Pink bars represent females and blue bars represent males. 25 30 35 40Temperature (˚C) Warm Detection Thresholds 30 35 40 45 50Temperature (˚C) Heat Pain Thresholds 35 40 45 50Temperature (˚C) Heat Pain Tolerance 20 25 30 35Temperature (˚C) Cool Detection Thresholds 0 10 20 30Temperature (˚C) Cold Pain Thresholds 0 60 120 180Time (s) Cold Pain Tolerance n = 89; 37 M, 52 F n = 89; 37 M, 52 F n = 91; 39 M, 52 F n = 91; 39 M, 52 F n = 81; 33 M, 48 F n = 91; 39 M, 52 F Less SensitiveMore Sensitive .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 14 WDT: Models for WDT are provided in Table S4a. The winning model for WDT was model 1 which included age, sex, and BMI (AIC = 243.0, R2 = 0.128, F3,85 = 4.17, p = 0.008). Sex was the only significant predictor in the model ( t = -2.83, p = 0.006, Table S4b), with female sex having lower WDT than males, indicating females were more sensitive to thermal sensations. FAAH SNP genotype, circulating FAAH substrates, and cannabis use were not associated with WDT (all p > 0.05). CDT: Models for CDT are provided in Table S5. There were no significant models for CDT, indicating that CDT is not associated with sex, FAAH SNP genotype, circulating levels of FAAH substrates or 2-AG, or cannabis-use (all p > 0.05). HPT: Models for HPT are provided in Table S 6. There were no significant models for HPT, indicating that HPT is not associated with sex, FAAH SNP genotype, circulating levels of FAAH substrates or 2-AG, and cannabis-use (all p > 0.05). CPT: Models for CPT are provided in Table S 7. There were no significant models for CPT, indicating that CPT is not associated with sex, FAAH SNP genotype, circulating levels of FAAH substrates or 2-AG, and cannabis-use (all p > 0.05). HPTol: Models for HPTol are provided in Table S8a. Model 2, which included age, sex, and BMI and FAAH allele (AIC = 227.48, R2 = 0.176, F4,76 = 4.05, p = 0.005) was the winning model. Sex was the only significant predictor in the model ( t = -2.87, p = 0.005, Table S8b), with female sex having lower HPTol than males. FAAH SNP genotype, circulating FAAH substrates, and cannabis use were not associated with HPT (all p > 0.05). CPTol: Models for CPTol are provided in Table S9. There were no significant models for CPTol, indicating that this measure is not associated with sex, FAAH SNP genotype, circulating levels of FAAH substrates or 2-AG, and cannabis-use (all p > 0.05). Mechanical QST measures Distributions of mechanical QST measures are presented in Figure 4. Figure 4. Distribution of mechanical QST measures. Each bar represents a single participant, and participants are ranked in ascending order. Pink bars represent females and blue bars represent males. 200 400 600 800 1000Pressure (kPa) Pressure Pain Threshold 0 20 40 60Difference in Pain Rating Temporal Summation of Pain Less SensitiveMore Sensitive n = 91; 39 M, 52 F n = 91; 39 M, 52 F .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 15 PPT: Models for PPT are provided in Table S10a. The winning model for PPT was Model 3, which included age, sex, BMI, FAAH SNP genotype, circulating FAAH substrates, and 2 -AG (AIC = 1202.65, R2 = 0.205, F6,84 = 3.60, p = 0.003). The circulating FAAH substrates variable was the only significant predictor in the model (t = 2.97, p = 0.004, Table S10b), indicating that those with higher levels of FAAH substrates had higher pain thresholds (i.e., less sensitive; Figure 5). FAAH SNP genotype, sex and cannabis use were not associated with PPT (all p > 0.05). Post-hoc partial correlation analyses controlling for age, sex, BMI, FAAH genotype and 2 -AG revealed that AEA and OEA were associated with PPT (AEA: r = .345, p = .00099; OEA: r = .300, p = 0.00494), but PEA was not associated (r = .133, p = .2234). TSP: Models for TSP are provided in Table S1 1. There were no significant models for TSP, indicating that TSP is not associated with sex, FAAH SNP genotype, circulating levels of FAAH substrates or 2-AG, and cannabis-use (all p > 0.05). Figure 5. Individual differences in pressure pain threshold are associated with circulating FAAH substrates. Values are adjusted for age, sex, body mass index, 2 -AG and FAAH genotype. The regression line is shown in red, and 95% confidence intervals are indicated with dashed blue lines.

Here, we report the relationship between baseline circulating levels of eCBs/NAEs and somatosensory sensitivity to thermal and mechanical stimuli in 91 healthy participants. Thermal measures (i.e., detection thresholds, pain thresholds, or pain tolerance) and TSP were not associated with baseline eCBs/NAEs. However, PPT did show a positive relationship with circulating FAAH substrates (i.e., a composite measure comprising AEA, OEA, and PEA). Post- hoc analyses revealed that the relationship was significant f or AEA and OEA, but not PEA. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 16 Furthermore, participants with either FAAH SNP variant ( CA or AA) did not display higher circulating levels of eCBs/NAEs compared to homozygote CC carriers. Additionally, there was no association found between cannabis use, eCBs and QST measures. Extensive preclinical work shows that cannabinoid receptors, their lipid ligands and metabolic enzymes lie at every relay site of the nociceptive neuraxis and the descending modulatory circuits [10,66,67]. Although the effects of FAAH inhibition are well documented in rodent chronic pain models, such effects have yet to be fully understood in acute pain. One study demonstrated that FAAH inhibition reversed mechanical hypersensitivity in a mouse model of acute thermal injury [68]. Another study demonstrated that pretreatment with a peripherally restricted FAAH inhibitor prevented the development of acute mechanical and thermal hypersensitivity in the carrageenan model of acute inflammation [77]. This is particularly interesting in the context of our study, given that we are measuring peripheral levels of eCBs/NAEs and their relationship with acute pain. As such, we measured baseline serum eCB/NAE levels that were not pharmacologically enhanced. Translating preclinical findings to humans has lagged, and existing studies are fragmented across small samples, heterogeneous pain assays, and various experimental pain models . For example, one study found no association between plasma eCB levels and PPT in either patients with chronic musculoskeletal pain or healthy controls; notably, the control group in this study included only 11 participants [82]. Similarly, there was no relationship observed between circulating eCB/NAE levels and PPT in female s with fibromyalgia [85] or in patients with neuromyelitis [71]. However, in another study, PPT in females with chronic widespread pain and females with chronic neck and shoulder pain was negatively correlated with microdialysate concentrations of PEA from the trapezius muscle at baseline [36]. Of note, the relationship between PPT and eCB/NAE in healthy controls was not investigated in these previous studies. Here, the association between higher FAAH-substrate levels and elevated PPT may reflect the anti -inflammatory or analgesic properties of endogenous AEA and other NAEs in deep tissues. During trapezius PPT testing, the algometer compresses the myofascial unit, activating high-threshold afferents in muscle tissue that contain nociceptive free nerve endings [59]. Indeed, it is possible that FAAH substrates could reduce PPT-induced activation and transmission in nociceptive pathways . Clinical evidence supports this mechanism: a meta-analysis of randomized controlled trials found that exogenous PEA confers modest analgesic relief across chronic -pain conditions [3]. As such, our data suggest that individual differences in baseline AEA and OEA are associated with PPT and thus may contribute to individual differences. In contrast to the above -mentioned studies, we did not observe a relationship between PEA and PPT ; the previous studies investigated changes in PEA, whereas ours focused on baseline PEA . Given the dynamic nature of eCBs/NAEs —i.e. that they are produced on demand [38,43,49,64,89], it is feasible that baseline measures of PEA are not as sensitive to individual differences in pain in healthy volunteers . Further studies are warranted, given the dearth of studies that have investigated eCBs and pain sensitivity in healthy participants. Together with the limited clinical data available, our study suggests that baseline circulating levels of eCBs/NAEs may not be a global predictor of pain sensitivity in healthy individuals. Rodent studies reveal sex differences in cannabinoid antinociception , with females generally showing enhanced sensitivity to cannabinoid -induced analgesia in various pain tests [22]. However, evidence regarding eCB tone is mixed, with some studies reporting that female rodents have higher AEA levels in discrete CNS regions [53], while others find higher levels in others find higher levels in other brain regions in males [12]. In our study, females displayed the .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 17 canonical lower WDT and HPTol, which has previously been reported in the literature [33]. However, we did not measure sex hormones, thereby not capturing previously reported cycle- dependent eCB fluctuations or progesterone –AEA interactions [15]. Interestingly, there was no association observed between circulating FAAH substrates and HPTol. This is surprising, given that AEA is also an agonist of TRPV1, the channel responsible for heat pain transduction and is activated by noxious heat (>43°C) [51]. However, baseline circulating levels of eCBs/NAEs may not be associated with point estimates of somatosensory and pain sensitivity in healthy individuals. Contrary to expectations and what has previously been reported in the literature [58,78], carriers of at least one FAAH C385A allele did not have higher circulating levels of AEA, PEA or OEA, and did not differ from CC homozygotes on any thermal or mechanical sensitivity tests. However, other studies with similar representation of the FAAH variant in their study population have also reported no statistically significant increase in circulating eCBs/NAEs [9]. In a study of 1,000 women undergoing surgery for breast cancer , individuals with the AA genotype for the C385A FAAH SNP were less sensitive to cold pain pre - operatively and reported less post - operative pain than individuals with CC and AC genotypes [14]. As our sample comprised only five participants with a FAAH SNP AA genotype, we could not test the contribution of this genotype on QST measures with any statistical confidence . Furthermore, as eCBs/NAEs are produced on demand, FAAH polymorphisms may influence pain primarily when the system is challenged by stress or injury, and this may be more pronounced in AA homozygotes; thus, larger samples enriched for AA homozygotes are warranted. Circulating eCBs are produced on -demand in response to stress [24,27,46,62], exercise [13,46], food presentation [35], inflammation and tissue injury [30,31,69], and time of day [40]. Circadian rhythms of circulating eCBs are complex and ligand -specific [40]: AEA shows a biphasic pattern peaking around 02:00 and 15:00 hours, with a nadir at 10:00 hours, while 2 -AG follows a monophasic rhythm, peaking at 12:00 –15:00 hours and dipping near 04:00 hours [40]. By sampling after a 12-hour fast and restricting testing to 09:00 –12:00 hours, we likely captured trough values for AEA and rising, but relatively low levels for 2-AG. Cannabis-use can also affect circulating levels of eCB s/NAEs in rodents [1]. In humans, evidence of differences in endocannabinoids in chronic users compared to non -users is mixed depending on the ligand, and the bio logical matrix sampled (e.g., cerebrospinal fluid, plasma, or serum) [9,48,54,60,61]. Studies investigating acute administration of exogenous cannabinoids in non-cannabis users have reported an increase in AEA and 2-AG within the following three hours of administration [84,87]. In our study, self -reported cannabis use was not associated with eCB levels or QST measures. Future studies should directly measure Δ⁹-THC metabolites in larger samples, across biological matrices to better determine the impact of cannabis usage on eCB/NAE levels. Several methodological limitations warrant consideration. Our cross -sectional design precludes causal inference regarding eCB-QST relationships. It is possible that a more comprehensive analysis that focuses solely on thermal pain sensitivity with multiple suprathreshsold stimuli at multiple sites might reveal an associated with eCBs/NAEs. Also, the unequal distribution of the FAAH genotypes in our sample limits our power to detect the effects of this genotype on QST responses. Additionally, the correlation between circulating serum concentrations and synaptic eCB levels within spinal or supraspinal pain circuits remains unknown [42]. Current non-invasive methodologies cannot adequately capture central nervous system eCB dynamics, as spinal tap to sample cerebrospinal fluid is associated with risks that are unjustifiable in healthy people. Furthermore, extant ligands for pharmacological brain imaging (i.e ., positron- .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 18 emission tomography), such as those developed for FAAH and CB 1 receptor, bind irreversibly [88]. Lastly, cannabis exposure assessment relied on self -report rather than toxicological confirmation, introducing potential reporting bias, and limited resolution. Future studies are required to determine: (i) whether d ynamic biochemistry is more sensitive to eCB/NAE -pain relationships—coupling serial blood or interstitial -fluid sampling to QST and intervention-based tasks could be more sensitive to eCB/NAE-pain relationships; and (ii) causal relationships between eCBs/NAEs and pain using p harmacological challenges (e.g. with FAAH inhibitors or CB1 receptor antagonists). Taken together, our data indicate that baseline circulating levels of eCBs/NAEs may not be a global predictor of experimental pain in healthy adults; nonetheless, higher circulating FAAH substrates are associated with higher PPT. These findings refine our mechanistic understanding of the human eCB system and its relationship to acute pain sensi tivity and highlight the importance of pain -modality test, dynamic ligand sampling, and individual biochemical profiling when designing eCB-based analgesic strategies. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 19

The authors have no conflicts of interest to declare. This work was supported by a Canadian Institutes of Health Research (CIHR) Project Grant awarded to M. M., D. P. F. and L. Y. A. (183703). M. M. holds a Canada Research Chair (Tier 2) in Pain NeuroImaging, and is supported by a University of Toronto Pain Scientist Award. L. Y. A. was supported in part by the Intramural Research Program of the National Institutes of Health (NIH; ZIA-AT000030). The contributions of the NIH author were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. S. B. was funded by a Government of Ireland Postgraduate Research Scholarship from the Irish Research Council (GOIPG/2019/3945). S. S. A. was supported by an Ontario Graduate Scholarship (OGS), a Natural Sciences and Engineering Research Council (NSERC) Canada Graduate Scholarship – Masters (CGS-M), and the University of Toronto’s Faculty of Dentistry Harron Fund Award. M. A. C. was supported by an NSERC CGS-M. R. T. was supported by University of Toronto’s Faculty of Dentistry Harron Fund Award, and an OGS. Dr. Liat Honigman, who is a co-author on this paper, was a beloved lab member and research associate in the Centre for Multimodal Sensorimotor and Pain Research (CMSPR) at the University of Toronto. She died on March 28, 2024. This, and many other projects in the lab would not have been possible without her expertise, guidance, and support. Liat was kind, smart, and a mentor to many trainees in the CMSPR. She held the highest standards for science, but believed in a compassionate approach to research. She is truly missed. Author Contributions: Conception of the study: MM, LYA and DPF Design of the Study: LH, IB and MM, DPF Data Acquisition: SAF, SB, NM, CS, MAC, RT Data Analysis: SSA, SAF, SB, KM Interpretation of the data: SAF, SSA, MM, LYA, DPF Drafting of manuscript: SAF, SSA, MM Reviewing and Editing of Manuscript and Final Approval: SAF, SSA, SB, KM, NM, RT, CS, MAC, IB, LYA, DPF, MM .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 20

[1] Ahmed F, Torrens A, Mahler SV, Ferlenghi F, Huestis MA, Piomelli D. A Sensitive Ultrahigh - Performance Liquid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Analysis of Phytocannabinoids and Endocannabinoids in Plasma and Brain. Cannabis Cannabinoid Res 9:371-385, 2024. [2] Amir Hamzah K, Toms LM, Kucharski N, Orr J, Turner NP, Hobson P, Nichols DS, Ney LJ. Sex - dimorphism in human serum endocannabinoid and n-acyl ethanolamine concentrations across the lifespan. Sci Rep 13:23059, 2023. [3] Artukoglu BB, Beyer C, Zuloff-Shani A, Brener E, Bloch MH. Efficacy of Palmitoylethanolamide for Pain: A Meta-Analysis. Pain Physician 20:353-362, 2017. [4] Ayoub LJ, McAndrews MP, Barnett A, Ho KCJ, Cioffi I, Moayedi M. Baseline Resting-State Functional Connectivity Determines Subsequent Pain Ratings to a Tonic Ecologically Valid Experimental Model of Orofacial Pain. PAIN 162:2397-2404, 2021. [5] Barry A, O'Halloran KD, McKenna JP, McCreary C, Harhen B, Kerr DM, Finn DP, Downer EJ. Plasma N-acylethanolamine and endocannabinoid levels in burning mouth syndrome: Potential role in disease pathogenesis. J Oral Pathol Med 47:440-442, 2018. [6] Beck AT, Steer RA, Brown GK. Manual for Beck Depression Inventory -II. Book Manual for Beck Depression Inventory-II. City: Psychological Corporation, 1996. [7] Benedetti F, Amanzio M, Rosato R, Blanchard C. Nonopioid placebo analgesia is mediated by CB1 cannabinoid receptors. Nat Med 17:1228-1230, 2011. [8] Blanton HL, Barnes RC, McHann MC, Bilbrey JA, Wilkerson JL, Guindon J. Sex differences and the endocannabinoid system in pain. Pharmacol Biochem Behav 202:173107, 2021. [9] Boachie N, Gaudette E, Bazinet RP, Lin L, Tyndale RF, Mansouri E, Huestis MA, Tong J, Le Foll B, Kish SJ, George TP, Boileau I. Circulating Endocannabinoids and N -Acylethanolamines in Individuals with Cannabis Use Disorder-Preliminary Findings. Brain Sci 13, 2023. [10] Bouchet CA, Ingram SL. Cannabinoids in the descending pain modulatory circuit: Role in inflammation. Pharmacol Ther 209:107495, 2020. [11] Bourke SL, Suarez EG, Islam B, Stephenson J, Finn DP, McHugh PC. Clinical measures in chronic neuropathic pain are related to the Kennedy and endocannabinoid pathways. Eur J Clin Invest 55:e14351, 2025. [12] Bradshaw HB, Rimmerman N, Krey JF, Walker JM. Sex and hormonal cycle differences in rat brain levels of pain-related cannabimimetic lipid mediators. Am J Physiol Regul Integr Comp Physiol 291:R349-358, 2006. [13] Brellenthin AG, Crombie KM, Hillard CJ, Koltyn KF. Endocannabinoid and Mood Responses to Exercise in Adults with Varying Activity Levels. Med Sci Sports Exerc 49:1688-1696, 2017. [14] Cajanus K, Holmstrom EJ, Wessman M, Anttila V, Kaunisto MA, Kalso E. Effect of endocannabinoid degradation on pain: role of FAAH polymorphisms in experimental and postoperative pain in women treated for breast cancer. Pain 157:361-369, 2016. [15] Centkowski SM, Lipoff JB. Inpatient dermatology consultations: Motivation and practice of requesting providers. J Am Acad Dermatol 77:1173-1174 e1173, 2017. [16] Chapman WP, Jones CM. Variations in Cutaneous and Visceral Pain Sensitivity in Normal Subjects. J Clin Invest 23:81-91, 1944. [17] Cheng JC, Erpelding N, Kucyi A, DeSouza DD, Davis KD. Individual Differences in Temporal Summation of Pain Reflect Pronociceptive and Antinociceptive Brain Structure and Function. J Neurosci 35:9689-9700, 2015. [18] Chiang KP, Gerber AL, Sipe JC, Cravatt BF. Reduced cellular expression and activity of the P129T mutant of human fatty acid amide hydrolase: evidence for a link between defects in the endocannabinoid system and problem drug use. Hum Mol Genet 13:2113-2119, 2004. [19] Coghill RC. Individual differences in the subjective experience of pain: new insights into mechanisms and models. Headache 50:1531-1535, 2010. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 21 [20] Coghill RC, McHaffie JG, Yen YF. Neural correlates of interindividual differences in the subjective experience of pain. ProcNatlAcadSciUSA 100:8538-8542, 2003. [21] Corcoran L, Roche M, Finn DP. The Role of the Brain's Endocannabinoid System in Pain and Its Modulation by Stress. Int Rev Neurobiol 125:203-255, 2015. [22] Craft RM, Wakley AA, Tsutsui KT, Laggart JD. Sex differences in cannabinoid 1 vs. cannabinoid 2 receptor-selective antagonism of antinociception produced by delta9 -tetrahydrocannabinol and CP55,940 in the rat. J Pharmacol Exp Ther 340:787-800, 2012. [23] Cravatt BF, Demarest K, Patricelli MP, Bracey MH, Giang DK, Martin BR, Lichtman AH. Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase. Proc Natl Acad Sci U S A 98:9371-9376, 2001. [24] Crombie KM, Leitzelar BN, Brellenthin AG, Hillard CJ, Koltyn KF. Loss of exercise - and stress- induced increases in circulating 2 -arachidonoylglycerol concentrations in adults with chronic PTSD. Biol Psychol 145:1-7, 2019. [25] Cuttler C, Spradlin A. Measuring cannabis consumption: Psychometric properties of the Daily Sessions, Frequency, Age of Onset, and Quantity of Cannabis Use Inventory (DFAQ -CU). PLoS One 12:e0178194, 2017. [26] Davis KD, Moayedi M. Central mechanisms of pain revealed through functional and structural MRI. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology 8:518-534, 2013. [27] Dlugos A, Childs E, Stuhr KL, Hillard CJ, de Wit H. Acute stress increases circulating anandamide and other N -acylethanolamines in healthy humans. Neuropsychopharmacology 37:2416 -2427, 2012. [28] Donvito G, Nass SR, Wilkerson JL, Curry ZA, Schurman LD, Kinsey SG, Lichtman AH. The Endogenous Cannabinoid System: A Budding Source of Targets for Treating Inflammatory and Neuropathic Pain. Neuropsychopharmacology 43:52-79, 2018. [29] Erpelding N, Moayedi M, Davis KD. Cortical thickness correlates of pain and temperature sensitivity. Pain 153:1602-1609, 2012. [30] Felton SJ, Kendall AC, Almaedani AF, Urquhart P, Webb AR, Kift R, Vail A, Nicolaou A, Rhodes LE. Serum endocannabinoids and N-acyl ethanolamines and the influence of simulated solar UVR exposure in humans in vivo. Photochem Photobiol Sci 16:564-574, 2017. [31] Fernandez-Rodriguez CM, Romero J, Petros TJ, Bradshaw H, Gasalla JM, Gutierrez ML, Lledo JL, Santander C, Fernandez TP, Tomas E, Cacho G, Walker JM. Circulating endogenous cannabinoid anandamide and portal, systemic and renal hemodynamics in cirrhosis. Liver Int 24:477-483, 2004. [32] Fillingim RB. Individual differences in pain: understanding the mosaic that makes pain personal. Pain 158 Suppl 1:S11-S18, 2017. [33] Folstein MF, Folstein SE, McHugh PR. "Mini -mental state". A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 12:189-198, 1975. [34] Fruhstorfer H, Lindblom U, Schmidt WC. Method for quantitative estimation of thermal thresholds in patients. J Neurol Neurosurg Psychiatry 39:1071-1075, 1976. [35] Gatta-Cherifi B, Matias I, Vallee M, Tabarin A, Marsicano G, Piazza PV, Cota D. Simultaneous postprandial deregulation of the orexigenic endocannabinoid anandamide and the anorexigenic peptide YY in obesity. Int J Obes (Lond) 36:880-885, 2012. [36] Ghafouri N, Ghafouri B, Larsson B, Stensson N, Fowler CJ, Gerdle B. Palmitoylethanolamide and stearoylethanolamide levels in the interstitium of the trapezius muscle of women with chronic widespread pain and chronic neck-shoulder pain correlate with pain intensity and sensitivity. Pain 154:1649-1658, 2013. [37] Glass M, Dragunow M, Faull RL. Cannabinoid receptors in the human brain: a detailed anatomical and quantitative autoradiographic study in the fetal, neonatal and adult human brain. Neuroscience 77:299-318, 1997. [38] Gregg LC, Jung KM, Spradley JM, Nyilas R, Suplita RL, 2nd, Zimmer A, Watanabe M, Mackie K, Katona I, Piomelli D, Hohmann AG. Activation of type 5 metabotropic glutamate receptors and .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 22 diacylglycerol lipase -alpha initiates 2 -arachidonoylglycerol formation and endocannabinoid - mediated analgesia. J Neurosci 32:9457-9468, 2012. [39] Guindon J, Hohmann AG. The endocannabinoid system and pain. CNS Neurol Disord Drug Targets 8:403-421, 2009. [40] Hanlon EC. Impact of circadian rhythmicity and sleep restriction on circulating endocannabinoid (eCB) N-arachidonoylethanolamine (anandamide). Psychoneuroendocrinology 111:104471, 2020. [41] Health Canada. Canadian Cannabis Survey 2024: Summary. In: H Canada editor. Book Canadian Cannabis Survey 2024: Summary. City: Government of Canada, 2024. [42] Hillard CJ. Circulating Endocannabinoids: From Whence Do They Come and Where are They Going? Neuropsychopharmacology 43:155-172, 2018. [43] Hohmann AG, Suplita RL, Bolton NM, Neely MH, Fegley D, Mangieri R, Krey JF, Walker JM, Holmes PV, Crystal JD, Duranti A, Tontini A, Mor M, Tarzia G, Piomelli D. An endocannabinoid mechanism for stress-induced analgesia. Nature 435:1108-1112, 2005. [44] Jacobson MR, Watts JJ, Boileau I, Tong J, Mizrahi R. A systematic review of phytocannabinoid exposure on the endocannabinoid system: Implications for psychosis. Eur Neuropsychopharmacol 29:330-348, 2019. [45] Jonsson KO, Vandevoorde S, Lambert DM, Tiger G, Fowler CJ. Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide. Br J Pharmacol 133:1263-1275, 2001. [46] Jurado-Fasoli L, Di X, Sanchez -Delgado G, Yang W, Osuna -Prieto FJ, Ortiz-Alvarez L, Krekels E, Harms AC, Hankemeier T, Schonke M, Aguilera CM, Llamas-Elvira JM, Kohler I, Rensen PCN, Ruiz JR, Martinez-Tellez B. Acute and long -term exercise differently modulate plasma levels of oxylipins, endocannabinoids, and their analogues in young sedentary adults: A sub -study and secondary analyses from the ACTIBATE randomized controlled -trial. EBioMedicine 85:104313, 2022. [47] Kelly KG, Cook T, Backonja MM. Pain ratings at the thresholds are necessary for interpretation of quantitative sensory testing. Muscle and Nerve 32:179-184, 2005. [48] Koethe D, Giuffrida A, Schreiber D, Hellmich M, Schultze -Lutter F, Ruhrmann S, Klosterkotter J, Piomelli D, Leweke FM. Anandamide elevation in cerebrospinal fluid in initial prodromal states of psychosis. Br J Psychiatry 194:371-372, 2009. [49] Kreitzer AC, Regehr WG. Retrograde inhibition of presynaptic calcium influx by endogenous cannabinoids at excitatory synapses onto Purkinje cells. Neuron 29:717-727, 2001. [50] Kroenke K, Spitzer RL, Williams JB. The PHQ -9: validity of a brief depression severity measure. J Gen Intern Med 16:606-613., 2001. [51] Kucyi A, Davis KD. The dynamic pain connectome. Trends Neurosci, 2014. [52] Lang -Illievich K, Klivinyi C, Lasser C, Brenna CTA, Szilagyi IS, Bornemann -Cimenti H. Palmitoylethanolamide in the Treatment of Chronic Pain: A Systematic Review and Meta-Analysis of Double-Blind Randomized Controlled Trials. Nutrients 15, 2023. [53] Levine A, Liktor -Busa E, Lipinski AA, Couture S, Balasubramanian S, Aicher SA, Langlais PR, Vanderah TW, Largent -Milnes TM. Sex differences in the expression of the endocannabinoid system within V1M cortex and PAG of Sprague Dawley rats. Biol Sex Differ 12:60, 2021. [54] Leweke FM, Giuffrida A, Koethe D, Schreiber D, Nolden BM, Kranaster L, Neatby MA, Schneider M, Gerth CW, Hellmich M, Klosterkotter J, Piomelli D. Anandamide levels in cerebrospinal fluid of first-episode schizophrenic patients: impact of cannabis use. Schizophr Res 94:29-36, 2007. [55] Long JZ, Li W, Booker L, Burston JJ, Kinsey SG, Schlosburg JE, Pavon FJ, Serrano AM, Selley DE, Parsons LH, Lichtman AH, Cravatt BF. Selective blockade of 2 -arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects. Nat Chem Biol 5:37-44, 2009. [56] Long JZ, Nomura DK, Vann RE, Walentiny DM, Booker L, Jin X, Burston JJ, Sim -Selley LJ, Lichtman AH, Wiley JL, Cravatt BF. Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo. Proc Natl Acad Sci U S A 106:20270- 20275, 2009. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 23 [57] Mattelaer N, Van der Schueren B, Van Oudenhove L, Weltens N, Vangoitsenhoven R. The circulating and central endocannabinoid system in obesity and weight loss. Int J Obes (Lond) 48:1363 -1382, 2024. [58] Mayo LM, Asratian A, Linde J, Holm L, Natt D, Augier G, Stensson N, Vecchiarelli HA, Balsevich G, Aukema RJ, Ghafouri B, Spagnolo PA, Lee FS, Hill MN, Heilig M. Protective effects of elevated anandamide on stress and fear-related behaviors: translational evidence from humans and mice. Mol Psychiatry 25:993-1005, 2020. [59] Mense S. Algesic agents exciting muscle nociceptors. Exp Brain Res 196:89-100, 2009. [60] Morgan CJ, Page E, Schaefer C, Chatten K, Manocha A, Gulati S, Curran HV, Brandner B, Leweke FM. Cerebrospinal fluid anandamide levels, cannabis use and psychotic -like symptoms. Br J Psychiatry 202:381-382, 2013. [61] Muhl D, Kathmann M, Hoyer C, Kranaster L, Hellmich M, Gerth CW, Faulhaber J, Schlicker E, Leweke FM. Increased CB2 mRNA and anandamide in human blood after cessation of cannabis abuse. Naunyn Schmiedebergs Arch Pharmacol 387:691-695, 2014. [62] Ney L, Stone C, Nichols D, Felmingham K, Bruno R, Matthews A. Endocannabinoid reactivity to acute stress: Investigation of the relationship between salivary and plasma levels. Biol Psychol 159:108022, 2021. [63] Nielsen CS, Staud R, Price DD. Individual differences in pain sensitivity: measurement, causation, and consequences. J Pain 10:231-237, 2009. [64] Ohno-Shosaku T, Maejima T, Kano M. Endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals. Neuron 29:729-738, 2001. [65] Okine BN, Madasu MK, McGowan F, Prendergast C, Gaspar JC, Harhen B, Roche M, Finn DP. N - palmitoylethanolamide in the anterior cingulate cortex attenuates inflammatory pain behaviour indirectly via a CB1 receptor-mediated mechanism. Pain 157:2687-2696, 2016. [66] Ossipov MH, Dussor GO, Porreca F. Central modulation of pain. J Clin Invest 120:3779-3787, 2010. [67] Palazzo E, Luongo L, Novellis V, Rossi F, Maione S. The Role of Cannabinoid Receptors in the Descending Modulation of Pain. Pharmaceuticals (Basel) 3:2661-2673, 2010. [68] Palmer JA, Higuera ES, Chang L, Chaplan SR. Fatty acid amide hydrolase inhibition enhances the anti-allodynic actions of endocannabinoids in a model of acute pain adapted for the mouse. Neuroscience 154:1554-1561, 2008. [69] Patsenker E, Sachse P, Chicca A, Gachet MS, Schneider V, Mattsson J, Lanz C, Worni M, de Gottardi A, Semmo M, Hampe J, Schafmayer C, Brenneisen R, Gertsch J, Stickel F, Semmo N. Elevated levels of endocannabinoids in chronic hepatitis C may modulate c ellular immune response and hepatic stellate cell activation. Int J Mol Sci 16:7057-7076, 2015. [70] Pecina M, Martinez-Jauand M, Hodgkinson C, Stohler CS, Goldman D, Zubieta JK. FAAH selectively influences placebo effects. Mol Psychiatry 19:385-391, 2014. [71] Pellkofer HL, Havla J, Hauer D, Schelling G, Azad SC, Kuempfel T, Magerl W, Huge V. The major brain endocannabinoid 2 -AG controls neuropathic pain and mechanical hyperalgesia in patients with neuromyelitis optica. PLoS One 8:e71500, 2013. [72] Pistis M, Muntoni AL. Roles of N -Acylethanolamines in Brain Functions and Neuropsychiatric Diseases. In: M Melis, editor. Endocannabinoids and Lipid Mediators in Brain Functions. Cham: Springer, 2017. [73] Raja SN, Carr DB, Cohen M, Finnerup NB, Flor H, Gibson S, Keefe FJ, Mogil JS, Ringkamp M, Sluka KA, Song XJ, Stevens B, Sullivan MD, Tutelman PR, Ushida T, Vader K. The revised International Association for the Study of Pain definition of pain: concepts, challenges, and compromises. Pain, 2020. [74] Rolke R, Baron R, Maier C, Tolle TR, Treede RD, Beyer A, Binder A, Birbaumer N, Birklein F, Botefur IC, Braune S, Flor H, Huge V, Klug R, Landwehrmeyer GB, Magerl W, Maihofner C, Rolko C, Schaub C, Scherens A, Sprenger T, Valet M, Wasserka B. Quantitative sensory testing in the German Research Network on Neuropathic Pain (DFNS): standardized protocol and reference values. Pain 123:231-243, 2006. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint Fatemi, Abssy et al. Contribution of eCBs to Pain Sensitivity 24 [75] Rolke R, Baron R, Maier C, Tölle TR, Treede RD, Beyer A, Binder A, Birbaumer N, Birklein F, Bötefür IC, Braune S, Flor H, Huge V, Klug R, Landwehrmeyer GB, Magerl W, Maihöfner C, Rolko C, Schaub C, Scherens A, Sprenger T, Valet M, Wasserka B. Quantitative sensory testing in the German Research Network on Neuropathic Pain (DFNS): standardized protocol and reference values. Pain 123:231-243, 2006. [76] Rolke R, Magerl W, Campbell KA, Schalber C, Caspari S, Birklein F, Treede RD. Quantitative sensory testing: a comprehensive protocol for clinical trials. EurJPain 10:77-88, 2006. [77] Sasso O, Bertorelli R, Bandiera T, Scarpelli R, Colombano G, Armirotti A, Moreno-Sanz G, Reggiani A, Piomelli D. Peripheral FAAH inhibition causes profound antinociception and protects against indomethacin-induced gastric lesions. Pharmacol Res 65:553-563, 2012. [78] Sipe JC, Chiang K, Gerber AL, Beutler E, Cravatt BF. A missense mutation in human fatty acid amide hydrolase associated with problem drug use. Proc Natl Acad Sci U S A 99:8394-8399, 2002. [79] Sipe JC, Scott TM, Murray S, Harismendy O, Simon GM, Cravatt BF, Waalen J. Biomarkers of endocannabinoid system activation in severe obesity. PLoS One 5:e8792, 2010. [80] Spagnolo PA, Ramchandani VA, Schwandt ML, Kwako LE, George DT, Mayo LM, Hillard CJ, Heilig M. FAAH Gene Variation Moderates Stress Response and Symptom Severity in Patients with Posttraumatic Stress Disorder and Comorbid Alcohol Dependence. Alcoholism , clinical and experimental research 40:2426-2434, 2016. [81] Starowicz K, Finn DP. Cannabinoids and Pain: Sites and Mechanisms of Action. Adv Pharmacol 80:437-475, 2017. [82] Stensson N, Grimby-Ekman A. Altered relationship between anandamide and glutamate in circulation after 30 min of arm cycling: A comparison of chronic pain subject with healthy controls. Mol Pain 15:1744806919898360, 2019. [83] The JASP Team. JASP (0.19.3) [computer software]. Retrieved from [84] Thieme U, Schelling G, Hauer D, Greif R, Dame T, Laubender RP, Bernhard W, Thieme D, Campolongo P, Theiler L. Quantification of anandamide and 2-arachidonoylglycerol plasma levels to examine potential influences of tetrahydrocannabinol application on the endocannabinoid system in humans. Drug Test Anal 6:17-23, 2014. [85] Vachon L, Costa T, Herz A. Desensitization of opioid-stimulated GTPase in neuroblastoma x glioma hybrid cells. Biochem Biophys Res Commun 128:1342-1349, 1985. [86] Wagner EJ. Sex differences in cannabinoid-regulated biology: A focus on energy homeostasis. Front Neuroendocrinol 40:101-109, 2016. [87] Walter C, Ferreiros N, Bishay P, Geisslinger G, Tegeder I, Lotsch J. Exogenous delta(9) - tetrahydrocannabinol influences circulating endogenous cannabinoids in humans. J Clin Psychopharmacol 33:699-705, 2013. [88] Wilson AA, Garcia A, Parkes J, Houle S, Tong J, Vasdev N. [11C]CURB: Evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography. Nucl Med Biol 38:247-253, 2011. [89] Wilson RI, Nicoll RA. Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses. Nature 410:588-592, 2001. [90] Woodhams SG, Chapman V, Finn DP, Hohmann AG, Neugebauer V. The cannabinoid system and pain. Neuropharmacology 124:105-120, 2017. [91] Yarnitsky D, Sprecher E. Thermal testing: normative data and repeatability for various test algorithms. J Neurol Sci 125:39-45, 1994. [92] Yarnitsky D, Sprecher E, Zaslansky R, Hemli JA. Heat pain thresholds: normative data and repeatability. Pain 60:329-332, 1995. [93] Zhao C, Ichimura A, Qian N, Iida T, Yamazaki D, Noma N, Asagiri M, Yamamoto K, Komazaki S, Sato C, Aoyama F, Sawaguchi A, Kakizawa S, Nishi M, Takeshima H. Mice lacking the intracellular cation channel TRIC -B have compromised collagen production and i mpaired bone mineralization. Sci Signal 9:ra49, 2016. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2025. ; https://doi.org/10.1101/2025.08.22.671762doi: bioRxiv preprint