Sprague–Dawley (SD) rat dams and pups (Envigo, Israel) and pups were housed in the animal facility of the School of Psychological Sciences at the University of Haifa. The rats were maintained on a 12:12 light/dark cycle with food and water provided ab libitum, in a polypropylene cages (59 × 28 × 20 cm), at a room temperature 22 ± 2°C. The day of birth was defined as postnatal day 0 (PND0). Pups were weaned on PND21 and arbitrarily assigned to same sex groups (4–5 animals per cage). This study focused on male rats, while female rats were used for the parallel experiments (Demaili et al., 2023). All experiments were approved by the University of Haifa Ethics and Animal Care Committee and appropriate measures were taken to minimize pain and discomfort (approval numbers: 741/2020, 765/2021).

For ELS, we used the established paradigm of maternal neglect (Avishai-Eliner et al., 2001) with modifications (Alteba et al., 2016, 2020, 2021; Portugalov et al., 2022). Briefly, from PND7 to PND14, dams and their pups were housed in a cage with limited bedding material, specifically a 1.2 cm layer of “Sunny Chips.” Before PND7 and after PND14 until weaning at PND21, dams and their litters were reared under standard conditions.

Male rats were arbitrarily assigned to one of two experimental groups:

Control (CON): The animals were not exposed to ELS and were group-housed together with their mother in a cage with adequate bedding material (7–9 cm layer) until weaning.

Early life stress (ELS): Animals in this group were exposed to the ELS paradigm as described above from PND7 to PND14.

Rats were exposed to a battery of behavioral tests in adulthood (PND 90–105) (Figure 1). Testing was performed under dim lighting (15–20 lx) between 13:00 and 16:00 h to reduce stress and facilitate more accurate observations of the animals’ behavior. We have consistently applied this lighting condition across behavioral experiments in previous studies (Portugalov et al., 2022; Bright and Akirav, 2023). Rats were arbitrarily assigned to the experimental groups (CON, n = 16; ELS, n = 14).

Animals from the respective experimental groups were sacrificed 3 days after the last behavioral test, on PND110. Brains were rapidly frozen in liquid nitrogen and stored at −80°C. For gene expression analysis, tissue from right and left medial prefrontal cortex (mPFC) tissue was dissected. The samples were obtained by punches from frontal sections at the level of the prefrontal cortex, corresponding to the following anatomical coordinates: Bregma + 2.70; Interaural + 11.70 (Paxinos and Watson, 2006). Collected samples included tissue from the anterior cingulate cortex (Acd, Cg1), prelimbic cortex (PrL) and infralimbic cortex (IL). Total RNA was extracted using the innuPREP RNA Mini Kit 2.0 (Analytik Jena, Berlin, Germany) following the manufacturer s instructions. Genomic DNA contaminants were removed using the innuPREP DNase I Digest Kit (Analytik Jena, Berlin, Germany). For analysis of gene expression, DNA-methylation, histone 3 global acetylation and histone 3 lysine 27 acetylation animals were arbitrarily assigned to the respective experimental groups (CON, n = 9; ELS, n = 10). Also for analysis of promoter specific histone 3 acetylation (nChIP) animals were arbitrarily assigned to the two experimental groups (CON, n = 6; ELS, n = 6).

One-step quantitative real-time PCR was performed using the QuantiNova Multiplex RT-PCR kit (Qiagen GmbH, Hilden, Germany) and Taqman gene expression assays (Life Technologies, Carlsbad, CA, USA) as described previously (Demaili et al., 2023). 20 ng of RNA was used as a starting material in a reaction volume of 20 μl. Commercially available rat Taqman probes were used for endocannabinoid receptor 1 (eCB1R, cnr1 gene) and fatty acid amide hydrolase (FAAH) as target genes (Rn00562880_m1 for cnr1 and Rn00577086_m1 for faah) and hypoxantine guanine phosphoribosyl transferase (HPRT) as reference gene (Rn01527840_m1). The qPCR reaction was carried out with real-time PCR cycler Rotor-Gene Q (Qiagen GmbH, Hilden, Germany). Cycling conditions were as follows: Denaturation, 95°C for 2 s.; Combined annealing/elongation 60°C for 30 s., total 40 cycles. Relative gene expression was calculated using the delta–delta Ct method (Livak and Schmittgen, 2001). All samples were run in two independent experimental replicates, with each gene assay performed in triplicates. CB1R and FAAH gene expression is presented as fold change relative to the respective mean value of the control group.

Deoxyribonucleic acid (DNA) methylation levels were determined using the bisulfite-converted DNA and pyrosequencing method, as described previously (Demaili et al., 2023; Kocamaz et al., 2022). In brief, genomic DNA was extracted from left mPFC using DNeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. EpiTect Bisulfite Kit (Qiagen GmbH, Hilden, Germany) and QIAcube (Qiagen GmbH, Hilden, Germany) were used for DNA bisulfite conversion and cleanup, respectively. Analyzed CpG sites within circumscribed DNA sequences in the promoter regions of the CB1R and FAAH genes were identified by using the National Center for Biotechnology Information (NCBI) databank. Primers covering 11 CpG sites in the promoter region of the CB1R gene were individually designed using PyroMark Assay Design Software (Qiagen GmbH, Hilden, Germany). For the FAAH gene, 2 primers covering 9 CpG sites within the promoter region were used, which are commercially available as PyroMark CpG assays (Qiagen). Pyrosequencing was performed with Pyromark Q96 ID (Qiagen GmbH, Hilden, Germany). For verification of methylation results, CpGenome rat methylated and unmethylated genomic DNA standards (Merck KGaA, Darmstadt, Germany) were used. DNA methylation levels were automatically analyzed using PyroQ CpG software (Qiagen GmbH, Hilden, Germany). The results are presented in two ways: (i) mean value of methylation levels across 11 analyzed CpG sites for CB1R and across 9 analyzed CpG sites for FAAH and (ii) CpG site specific methylation levels at the analyzed individual CpG sites.

Global histone 3 acetylation (H3ac) and histone 3 lysine 27 acetylation (H3K27ac) levels were determined by quantitative western blot (WB) analysis, as described previously (Köhler et al., 2019). In short, tissue samples were homogenized in extraction (lysis) buffer (0.1 M Tris/HCL pH 8.0; 0.01 M EDTA; 10% SDS); 1× Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). After cell lysis, we continued with sonification (ultrasonic vibration), to isolate nuclei. Protein concentration was measured using the Bio-Rad DC™ Universal Protein Assay Kit II (Bio-Rad, Hercules, CA, USA), and was in a range between 10 and 20 μg/μL. Gel electrophoresis was performed using 30 μg of protein with loading buffer and Bio-Rad Tris-Glycine Mini-PROTEAN TGX Precast Gels (Bio-Rad, Hercules, CA, USA). Size marker PeqGOLD Protein Marker V (PeqLab/VWR International GmbH, Darmstadt, Germany) was used to determine the protein molecular weight. After gel electrophoresis, the samples were blotted onto a nitrocellulose membrane (GE Healthcare, Chalfont St Giles, UK). The blots were blocked with RotiBlock BSA blocking reagent (Carl Roth, Karlstuhe, Germany), followed by overnight incubation with primary antibodies: anti-acetyl H3 (#06-599, 1:10,000; Merck Millipore, Billerica, MA, USA), anti-acetyl H3K27 (#39133, 1:2,000; Active Motif, Carlsbad, CA, USA) and antihypoxanthine phosphoribosyltransferase I (HPRT1, #PA-522281, 1:500; Thermo Fisher Scientific, Waltham, MA, USA) at 4°C. The antibodies specific for histone 3 have been used in our previous study (Köhler et al., 2019). The next day, blots were incubated for 1-h at room temperature with a secondary antibody (horseradish peroxidase conjugated anti-rabbit; #12–348; 1:4,000; Merck Millipore, Billerica, MA, USA). Horseradish peroxidase was detected with Luminata Crescendo Western HRP substrate (Merck Millipore, Billerica, MA, USA). Syngene G:Box system and Gene Tools software (Syngene Europe, Cambridge, UK) were used for signal detection and data analysis, respectively. H3ac/HPRT and H3K27/HPRT ratio is presented as fold change relative to the mean value of the respective control group.

Histone 3 acetylation on CB1R and FAAH gene promoters was quantified by native chromatin immunoprecipitation (nChIP). NChIP protocol was performed following an established protocol (Xie et al., 2013; Köhler et al., 2019) with specific modifications for rat brain tissue (Donovan and Lichota, 2014). In short, nuclei were extracted using a glass homogenizer and a combination of extraction buffers: buffer 1; 0.4 M sucrose, 10 mM Tris–HCl pH = 7.9, 5 mM sodium butyrate, protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA), buffer 2; 0.25 M sucrose, 10 mM Tris–HCl pH = 7.9, 5 mM sodium butyrate, protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA), 1% Triton X-100, 10 mM MgCl₂. Digestion step was done with digestion buffer; 0.32 M sucrose, 50 mM Tris–HCl pH = 7.5, 5 mM sodium butyrate, protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA), 4 mM MgCl₂, 1 mM CaCl₂ and with 30 U MNase (Thermo Fisher Scientific, Waltham, MA, USA). The enzymatic digestion was stopped by adding 0.5 M EDTA. Sample was centrifuged at 11,600 g, 4°C for 5 min. Supernatant was divided and mixed with anti-acetyl-H3 antibody (#06–599, Merck Millipore, Billerica, MA, USA), then incubated overnight at 4°C with constant rotation. For immunoprecipitation protein A/G Magnetic Beads were used (Thermo Fisher Scientific, Waltham, MA, USA). The beads and antibody-sample mixture were incubated at 4°C for 3 h while rotating. Beads were then collected on magnet and washed with following buffers for 10 min each: Low salt wash buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 7.9), high salt wash buffer (500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 7.9) and 3× TE buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA). After washing steps, samples were eluted in freshy prepared elution buffer (1% SDS, 0.1 M NaHCO₃), for 2 × 15 min. After the last elution step, antibody-bound samples and inputs were incubated with 0.5 M EDTA, 1 M Tris–HCl pH 6.8 and 20 μg Proteinase K (Thermo Fisher Scientific, Waltham, MA, USA) for 3 h at 45°C in a thermomixer. After proteinase K treatment, DNA was extracted with phenol/chloroform/isoamylalcohol (Carl Roth GmbH, Karlsruhe, Germany) and subjected to DNA purification, by MinElute Reaction CleanUp Kit (Qiagen GmbH, Hilden, Germany). The results were analysed by ChIP quantitative real-time PCR, using the QuantiNova Multiplex RT-PCR kit (Qiagen GmbH, Hilden, Germany) and custom-made TaqMan assays targeting CB1R (FAM-coupled) and FAAH (VIC-coupled; Thermo Fisher Scientific, Waltham, MA, USA). For both CON and ELS groups, 6 animals were used per group. All the samples were run in three independent experiments and in duplicates within one experiment. Fold enrichment in histone H3 acetylation on the CB1R and FAAH gene promoter regions was calculated with formula: 2^{−[Ct(H3ac) − Ct (input)]}.

Differences between the experimental groups for individual behavioral tests, gene expression, mean DNA methylation, CpG site specific methylation, H3ac and H3K27ac were analysed using a two-tailed, unpaired t-test analysis. Behavioral profiling was conducted as described above and the distribution of affected versus unaffected populations was calculated by using Pearson’s chi-squared test. All statistical calculations were performed using GraphPad Prism 8 (San Diego, CA, USA), with the exception of behavioral profiling, which was analyzed using SPSS 27 (IBM, Chicago, IL, USA). All values were presented as mean value ± SD. p < 0.05 was considered statistically significant. No sample size calculation was performed. Number of subjects for behavioral analysis as well as for molecular changes was estimated based on previous studies from our groups (Demaili et al., 2023; Portugalov et al., 2022; Kocamaz et al., 2022). All animals for the respective methodological approaches were used for statistical analysis and thus no outlier test were performed.

Various ELS paradigms have been applied in preclinical studies to study the immediate and long-term impact on the behavioral, and on the molecular and physiological level in the brain. One established paradigm involves rearing animals with reduced bedding material during the second postnatal week, resulting in abnormal maternal care characterised by fragmented and unpredictable maternal behavior as well as heightened sensitivity to stress in offspring and impaired hipocampus-mediated spatial learning and dendritic atrophy in the hippocampal formation (Gilles et al., 1996; Avishai-Eliner et al., 2001; Ivy et al., 2008; Ivy et al., 2010). The limited bedding paradigm offers a controlled environment to study the long-term effects of ELS, providing valuable data with direct implications for human research and treatment approaches. The persistence of social and emotional deficits in ELS-exposed rats throughout their lifespan suggests that early interventions may be crucial in preventing long-term mental health consequences in humans. By understanding how ELS influences behavior and brain development in animal models, researchers can develop targeted interventions to mitigate these effects, potentially reducing the incidence of depression, social anxiety, and related disorders in humans (Molet et al., 2014; Baram et al., 2012; Rice et al., 2008; Joshi and Onaivi, 2021). In the current study we applied a slightly modified maternal neglect paradigm in rats that has previously been shown to lead to impaired sociability, anxiety-like and depression-like behavior in adolescent and adult rats (Alteba et al., 2016, 2020, 2021; Portugalov et al., 2022; Demaili et al., 2023).

Along this line, the present study provides further evidence that ELS-exposed male rats develop anhedonic behaviour lasting into adulthood as measured in the saccharine preference test. Anhedonia, the inability or loss of pleasure in a wide range of activities, is among the core symptoms to diagnose a major depressive disorder in humans according to the DSM-5 (American Psychiatric Association, 2013; Hamilton, 1967). Anhedonia can be divided into subcomponents, consummatory anhedonia, which is the emotional “liking” component reflected by reduced hedonic response and motivational anhedonia, the “wanting” component, reflected by loss of interest or motivation (Treadway and Zald, 2011). Since in animal models these specific diagnostic criteria cannot be applied completely, a number of behavioral tests have been developed to test for specific aspects of depressive-like behavior that reflect symptoms of the mental disorder in humans (Gencturk and Unal, 2024). In our study, anhedonia was reflected by reduced preference for saccharine. The saccharine or sucrose preference test is one of the most common tests for assessing anhedonia and covers the consummatory component, but to some extend also motivational aspects observed in our previous studies, where the results from forced swim test (FST) unveiled symptoms of despair and helplessness in ELS-exposed adult male and female rats (Portugalov et al., 2022; Alteba et al., 2020).

In the present study, we also observed that adult ELS-exposed male rats showed disturbed social behavior, as indicated by results of the social preference test, where ELS-exposed rats spent less time with social partner. This observation replicates findings from our previous studies, where we have found reduced sociability of ELS exposed male and female rats, in adolescence and also in adulthood (Demaili et al., 2023; Alteba et al., 2020, 2021). This dysfunctional social behavior was detectable as early as PND 20, and appears to persist throughout lifespan (Raineki et al., 2012, 2015). Impairments in social behavior or interaction can be a result of the combination of symptoms for depressive disorders according to the DSM-5 (American Psychiatric Association, 2013). In addition, the observed ELS-induced impairment of social behavior might be indicative of symptoms of autistic spectrum disorder, in which the endocannabinoid system has been shown to play a major role (Joshi and Onaivi, 2021).

Interestingly, the observed deficits in socio-emotional behavior were not paralleled by symptoms of enhanced anxiety in the open field test (OF), which is in line with findings from our previous study applying the same ELS-paradigm (Alteba et al., 2020) and a study, which reported that ELS-induced anhedonia was not accompanied by measures of anxiety (Molet et al., 2016). In contrast, the present results of the EPM test revealed reduced anxiety in ELS-exposed males. A similar finding was observed in a study, using maternal separation as ELS paradigm, reporting reduced anxiety in adolescent males exposed to ELS (Wang et al., 2015). With regard to the interpretation of these results it remains to be clarified whether the reduced anxiety symptoms observed in ELS-exposed animals may be related to increased risk-taking behavior and/or may indicate positive adaptations to early adverse events leading to situation-specific resilience.

Numerous studies have shown that adverse environmental challenges in early life, in particularly stress, influence the functional development of the endocannabinoid system in different brain areas (Marco et al., 2014; Moussa-Tooks et al., 2020; Hill et al., 2019; Romano-López et al., 2016; Amancio-Belmont et al., 2020). Consistent with these findings, our study provides evidence that ELS-induced behavioral changes are paralleled by altered endocannabinoid function. For both, CB1R and FAAH mRNA, expression levels in the mPFC were reduced in adult ELS-exposed males compared to unstressed control animals. These results are in accordance with previous observations (Alteba et al., 2021), where we reported that adult ELS-exposed male rats showed a decrease of CB1R expression in the mPFC-hippocampal circuit, correlating with the behavioral outcome, including despair-like behavior. Other studies using maternal separation as the ELS paradigm also reported reduced CB1R binding in the mPFC of male rats during adolescence and adulthood (Hill et al., 2019; Goldstein Ferber et al., 2021).

There is considerable evidence that eCB signaling is critically involved in the regulation of stress responses. Reduced activity of the main regulatory elements of the endocannabinoid system is related to dysfunctional stress regulation, fear responses and anxiety symptoms (Hillard, 2014; Jenniches et al., 2016; Patel et al., 2017). Regarding the functional/physiological interpretation of our molecular data, it is important to note that CB1R expression is particularly prominent in brain areas, especially the PFC, that are involved in anxiety and stress regulation, behavioral flexibility and adaptation as well as in learning and memory functions. ECS is a homeostatic system; therefore it is safe to suggest that CB1R and FAAH downregulation in the mPFC leads to dysregulated endocannabinoid signalling in this brain area and a consequentially higher risk for the development of mood disorders and psychiatric diseases. The altered behavior can potentially be rescued with a pharmacological treatment approach, as we have previously shown (Mizrachi Zer-Aviv et al., 2022). In this study, we had male rats exposed to a single foot shock. Rats performed poorly in the behavioral tests battery, including low saccharine consumption and higher immobility time in the FST, as well as lower scores in social preference and recognition tests. Pharmacological intervention with FAAH inhibitor, URB597, has restored these behaviors, specifically social behavior and depressive-like symptoms. Similarly, for ELS males, we have found that URB597 applied in adolescence restored ELS-induced freezing in FST and social recognition (Portugalov et al., 2022). Both of our previous studies imply ECS involvement in depressive-like behavior and social disorders and suggest potential future therapy directions.

CB1R is also involved in synaptic plasticity mechanisms such as long-term potentiation (LTP; Abush and Akirav, 2013) and long-term depression (LTD; Castillo et al., 2012). Presynaptic CB1Rs are retrogradely activated by postsynaptically released 2-AG or AEA and thereby can inhibit the release of GABA (resulting in disinhibition) and/or glutamate (reducing excitatory activity), thus modulating the essential excitatory-inhibitory balance in neuronal networks. As a consequence, disturbed CB1R signaling may hinder the mentioned plasticity processes, leading to difficulties in adapting to new environmental challenges. However, the methodology used in this study does not allow a cellular or synaptic resolution and thus it can only be speculated that the observed ELS-induced downregulation of CB1R as well as FAAH expression in the mPFC might indicate a dysbalance of excitation/inhibition in this brain region, potentially affecting short- and long-term synaptic plasticity (Scheyer et al., 2023). Moreover, it is tempting to speculate that the ELS-induced downregulation of FAAH expression in the mPFC, observed in the ELS-exposed males, may be associated with the measured symptoms of reduced anxiety. This hypothesis is supported by pharmacological studies, which show that suppression of FAAH activity exerts anxiolytic effects (Fidelman et al., 2018; Segev et al., 2014; Griebel et al., 2018; Marco et al., 2015; Haller et al., 2009; Scherma et al., 2008). Since CB1Rs are located not only on presynaptic terminals of neurons but also on astrocytes (Yin et al., 2019; Scheyer et al., 2023), their downregulation may also distrupt astrocytic functions, including the modulation of synaptic transmission and neuroinflammatory responses. On the other hand, FAAH, an enzyme responsible for degrading AEA, and a negative regulator of ECS signalling, is located in postsynaptic structures as well as in astrocytes and microglia. Reduced FAAH expression could impact neuroinflammatory pathways, which are implicated in various psychiatric disorders. Taken together, the concurrent reduction of both CB1R and FAAH suggests a complex dysregulation of the endocannabinoid system, which may impair synaptic plasticity and contribute to maladaptive stress responses and the development of mental disorders.

In this study we focused on the long-term effects of ELS exposure on adult male rats, whereas our previous study focused on female rats (Demaili et al., 2023). Comparing the results for male and female rats behavioral profiling revealed that the percentage of affected/vulnerable subjects was higher in ELS-females (50% affected) than in ELS-males (28% affected). This is consistent with multiple studies, reporting that females have an increased risk for the development of mental disorders following adverse experiences (Whitaker et al., 2021; Rubinow and Schmidt, 2019; Goodwill et al., 2019). Moreover, our two studies revealed that males and females appear to develop slightly different phenotypes regarding anxiety behavior. While males in the present study displayed reduced anxiety levels without motor hyperactivity, female rats in the previous study showed higher motor activity (perhaps indicative of hyperactivity symptoms) but no changes in anxiety levels (Demaili et al., 2023). In contrast, no sex-differences were observed in depressive-like and social behavior, as both males (present study) and females (Demaili et al., 2023) displayed similar symptoms of anhedonia and reduced social interest. Regarding the detected ELS-induced changes in the endocannabinoid system we found opposing sex-specific effects. The adult males in the present study exibited a downregulation of CB1R and FAAH expression in the mPFC, whereas adult ELS females showed an upregulation of CB1R and no changes of FAAH expression in the mPFC (Demaili et al., 2023). This suggests a sexual dimorphism in the developmental trajectories and maturation of the ECS as a potential explanation for the observed sex-specific effects. Postnatally, the ECS develops at different rates in the male and female rat brain. There is a high number of CB1Rs until the pruning process in late adolescence and adulthood, with females having the highest numbers of CB1R at PND30 and males at PND40 (Rodríguez de Fonseca et al., 1993). ECS dimorphism is particularly evident in the PFC, as seen in another rat study (Bernabeu et al., 2020). Endocannabinoid-mediated maturational trajectories were active in juvenile females, but lacking in males, where they emerged later in puberty.

Increasing evidence reveals that the perinatal adverse experiences influence brain and behavior through both transient and permanent epigenetic mechanisms (Bock, 2024; Szyf, 2021; Bock et al., 2015; Braun et al., 2020; Alyamani and Murgatroyd, 2018; Li et al., 2020; Uchida et al., 2010). Epigenetic modification represents a crucial regulator for the fine-tuning of gene expression and the classical view is that enhanced DNA-methylation (hypermethylation) leads to decreased or even silenced gene expression, whereas increased histone acetylation is associated with open, transcriptionally active chromatin resulting in elevated gene expression (Gräff and Mansuy, 2008; Allis and Jenuwein, 2016; Greenberg and Bourc'his, 2019). We hypothesized that ELS “reprograms” the expression of the CB1R and FAAH genes, and that the changes observed in ELS-exposed adult males are due to epigenetic alterations. While we did not detect significant changes in DNA methylation within the promoter regions of the CB1R and FAAH genes, we observed significantly lower global histone H3 acetylation levels in ELS-exposed males compared to unstressed, control males. This supports our working hypothesis that the observed ELS-induced downregulation of CB1R and FAAH gene expression is epigenetically regulated.

This interpretation is corroborated by other studies that show post-translational histone acetylation regulates ELS-induced changes of gene transcription, replication and DNA structure. For example, in one of our previous studies in mice (Xie et al., 2013), where 3 days of maternal separation was applied as mild ELS exposure, we observed a rapid increase in acetylation levels of H3 and H4 immediately after the last separation period on PND 16. This increased histone acetylation was directly associated with increased expression levels of the synaptic plasticity genes Arc and Egr1 in the hippocampus.

The effect on histone acetylation appears to be transient. In contrast to the immediate increase, applying the same stress paradigm, we found a decrease in H3 acetylation levels in the mPFC of adult ELS-males detected in the present study. Similarly, a study that applied a stronger stressor, chronic unpredictable stress, reported that male mice had lower histone 3 lysine 9 acetylation (H3K9ac) levels associated with CB1R gene expression (Lomazzo et al., 2017).

However, it should be noted that nChIP analysis did not detect significant ELS-induced promoter-specific changes of H3 acetylation within the promoter regions of the CB1R and FAAH genes. We have taken into consideration that only circumscribed sequences within the promoter regions were analyzed and that regulatory elements controlling the interaction between histone H3 acetylation and chromatin might be located in other parts of the promoter or even at other regions such as enhancer (Engelen et al., 2015; Ji et al., 2015; Chen et al., 2019).