Endocannabinoid system genes regulation in the effects evoked by social isolation in rats

doi:10.21203/rs.3.rs-3586582/v1

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Endocannabinoid system genes regulation in the effects evoked by social isolation in rats

https://doi.org/10.21203/rs.3.rs-3586582/v1

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published 18 Feb, 2024

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Social isolation is a state of lack of social connections, involving the modulation of different molecular signalling cascades and associated with high risk of mental health issues. To investigate if and how gene expression is modulated by social experience at the central level, we analyzed the effects of 5 weeks of social isolation in rats focusing on endocannabinoid system genes transcription in key brain regions involved in emotional control. We observed selective reduction in mRNA levels for fatty acid amide hydrolase (Faah) and cannabinoid receptor type 1 (Cnr1) genes in the amygdala complex and of Cnr1 in the prefrontal cortex of socially isolated rats when compared to controls, and these changes appears to be partially driven by trimethylation of Lysine 27 and acetylation of Lysine 9 at Histone 3. The alterations of Cnr1 transcriptional regulation result also directly correlated with those of oxytocin receptor gene. We here suggest that to counteract the effects of SI, it is of relevance to restore the endocannabinoid system homeostasis via the use of environmental triggers able to revert those epigenetic mechanisms accounting for the alterations observed.

Social isolation

Endocannabinoid system

Epigenetics

Endocannabinoid-Oxytocinergic systems interplay

Rats

Social isolation (SI), defined as the lack of social relationships [1], has been linked to higher risks for different physical and mental conditions, due in part to bad habits like smoking, high alcohol consumption, unbalanced diet, and physical inactivity [2–4]. Moreover, it may be the cause for premature mortality, as well as for anxiety and depression [5–7]. Loneliness and minimal contacts or interactions with others are indexes of SI, however biological measures of SI have not yet been defined due to the complex nature of social interactions. Because of the isolation many experienced recently during the COVID-19 pandemic, it seems of relevance and timely to better understand the exact mechanisms underlying isolation-induced behaviors.

It is well-known that life and social experiences may induce modulation of gene expression at the brain level [8–10] with consequences on behavioral responses. In fact, gene expression in different species can be modulated by SI [11–13].

Accumulating evidence suggest that epigenetic mechanisms, regulating changes in genes expression without any modification of the DNA sequence, modulate social behaviors [14–19]. How these changes in gene expression might be affected by life stressors, such as SI, is today poorly understood [13, 20, 21].

To study the effects of SI on mental health a common tool is the use of rat models exposed to SI at early-life stages [22]. Deprivation of social interactions induces in rats behavioral and neurochemical alterations mimicking features of different human psychopathologies [23, 24], and drug addiction [25–29]. A significant body of evidence shows a key role in the social reward system for the neuromodulator peptide Oxytocin (OXT) and its receptor (OXTR) expressed in brain regions [30–35] where we recently showed, using a rat model of SI, alterations in receptor mRNA levels [36]. In this work, we want to evaluate how and if the alterations evoked by SI on OXTR might interact with potential modulation of the endocannabinoid system (ECS), which signalling has been already observed to be OXT mediated [37]. Studies support a distinct role for the ECS, composed of cannabinoid receptors, their ligands (the endocannabinoids) and the enzymes needed for their metabolism, in the control of social behavior resulting dysregulated in stressful conditions [38, 39] as well as in social play behaviors [40] and social anxiety [41, 42]. It has been recently reported that subjects in self-isolation due to COVID-19 tend to use 20% more cannabis compared to those who did not [43]. Moreover, social anxiety has been associated with frequent cannabis use [44] that evokes several neuroadaptations in the ECS [45].

We here report data on the modulation of ECS genes expression evoked by the exposure to SI in rats analyzing different brain regions, namely prefrontal cortex (Pfc), amygdala complex (Amy), hypothalamus (Hyp), nucleus accumbens (NAc) and hippocampus (Hip). In particular, we investigated the role of epigenetic mechanisms, playing a key role in the transcriptional regulatory networks that social behavior is able to establish and modulate [14–19].

Animal model of social isolation

Experiments were carried out on a rat model previously described [36], using Wistar female pregnant rats singly housed under controlled conditions. In the present study, after weaning at PND 21, rats have been housed for five weeks (from PND 21 to PND 54) individually (ISO group) or in groups of three per cage (CTRL group). This isolation period was chosen as it coincides with the initiation and maximal expression of play behavior, that is the most characteristic social behavior displayed by developing mammals, including rats [46]. In rats, play behavior emerges around PND 16 and increases until it peaks on ~ PND30 [47, 48]. Rats deprived of this experience during this juvenile period show behavioral abnormalities during adulthood in the socio-cognitive domains, and these are associated with neuronal alterations in the prefrontal cortex [49]. Sample size for each group (N = 8) was based on our previous experiments, and power calculations were performed using G*Power version 3.1 [50]. Experimental set-up was performed according ARRIVE guidelines [51, 52], and the European Community Directive 2010/63/EU. Behavioral tests have been performed on animals from both experimental groups at PNDs 55–58 [36]. At PND 61, after testing, rat brains were collected and frozen immediately.

Molecular studies

Nucleic acids and chromatin were extracted from the following brain regions: Pfc, Amy, Hyp, NAc and Hip, as previously described [53], to analyze mRNA levels, DNA methylation mark and histone modifications.

Gene expression analysis by Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)

RNA (1 µg) was converted into cDNA by SensiFAST™ cDNA Synthesis kit (Bioline Reagents, London, UK). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using SensiFAST™ SYBR®Lo-ROX Kit (Bioline Reagents, London, UK) on a DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research). mRNA relative abundance was first normalized to two endogenous housekeeping genes Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and β-actin (β-Act), then was calculated by the Delta-Delta Ct (ΔΔCt) method and converted to relative expression ratio 2^(−ΔΔCt) [54]. Sequences of the primers used are reported in Table S1.

DNA methylation analysis

To analyze methylation levels of Cnr1 (coding for the cannabinoid receptor 1, Cb1) and Faah (coding for the fatty acid amide hydrolase, Faah), genomic DNA was isolated from selected rat brain regions, as previously described [53].

Briefly, 500 ng of purified DNA was subjected to bisulfite conversion using the EZ DNA Methylation-GoldTM Kit (Zymo Research, Orange, CA, USA) as previously described [55]. CpG islands prediction at Cnr1 and Faah gene promoters was performed with MethPrimer [56]. The DNA methylation status of 8 CpG sites within the Cnr1 CpG island and 5 CpG sites in the Faah island were analyzed using a pyrosequencing assay [57].

The CpG islands under study at Cnr1 and Faah rat genes are shown in Figs. 1a and 3a. Cnr1 CpG methylation custom assay was designed through PyroMark Assay Design Software 2.0 (Qiagen, Hilden, Germany), while Faah CpG methylation assay was purchased from Qiagen (Rn_Faah_01_PM PyroMark CpG assay, PM00559699, Qiagen, Hilden, Germany). Bisulfited DNA was amplified with the PyroMark PCR Kit (Qiagen, Hilden, Germany) using a biotin-labelled primer, according to the manufacturer’s protocol. PCR conditions were as follows: after an initial step at 95°C for 15 min, samples were subjected to 45 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, and, finally, 72°C for 10 min. Specificity of PCR products was then verified by gel electrophoresis (1.8% agarose). Sequencing of the amplification products was performed on a PyroMark Q48 Autoprep using PyroMark Q48 Advanced Reagents (Qiagen, Hilden, Germany), following the manufacturer’s recommendations. DNA methylation level was assessed through the PyroMark Q48 Autoprep 2.4.2 software, calculating, for each CpG site, the methylation percentage: mC / (mC + C) %, where mC = number of methylated cytosines, C = number of unmethylated cytosines, allowing quantitative comparisons. Methylation percentage levels were calculated for every CpG site and for the average of all the CpG sites under study, for both Cnr1 and Faah genes. Analyzed sequences, primers and assays used for the analyses are reported in Table S2.

ChIP-qPCR analysis

Chromatin immunoprecipitation was performed according to Dahl and Collas protocol [58] with minor modifications. Briefly, brain tissues were subjected to DNA-protein crosslinking adding formaldehyde 1% (v/v) (Sigma-Aldrich, Darmstadt, Germany) in phosphate buffer saline containing a broad-range protease inhibitor cocktail (PIC) (Sigma-Aldrich, Darmstadt, Germany) and incubating for 10 min at room temperature. Reaction was stopped adding glycine at 125 mM final concentration, incubating for 5 min at room temperature. Tissues were washed three times and lysed with 120 µl of lysis buffer at room temperature (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS and PIC (Sigma-Aldrich, Darmstadt, Germany)). Chromatin fragmentation was performed by an ultrasonic homogenizer (Bandelin Sonoplus UW 2070, ProfiLab24 GmbH, Berlin, Germany) for 6 x 30 s cycles on ice at 30% power, allowing 15 s pause between each pulsing session to produce 700 − 400 bp fragments. A centrifugation step at 12000 x g for 10 min at 4°C was performed, and supernatants transferred in frosted tubes. DNA fragments were then tested on 1.2% agarose gel. A slurry of A/G paramagnetic beads (Dynabeads® Protein A, Invitrogen, Carlsbad, CA, USA) was prepared removing beads buffer after magnetic capture and washing beads twice in RIPA buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA, 1% Triton-X 100, 0.1% SDS, 0.1% Na-deoxycolate, 140 mM NaCl). 10 µl of pre-washed A/G-bead slurry were incubated separately with 1 µg of Anti-Acetyl-Histone3-Lysine-9 (Anti-H3K9Ac) (PA5-117092 ThermoFisher, MA, USA) and Anti-Trimethyl-Histone-3-Lysine-27 (Anti-H3K27Me3) (PA5-114540 ThermoFisher, MA, USA) antibodies at 4°C overnight. Chromatin was 10-fold diluted in RIPA buffer + PIC and 100 µl were transferred in 1.5 ml tubes containing antibody-coated A/G beads and were incubated on a rotator at 40 rpm at 4°C for 4 h. Aliquots of diluted chromatin were removed and kept separately as input (positive internal control). Three wash steps were performed in RIPA buffer (4 min on a rotator at 40 rpm at 4°C). Another wash step was performed in Tris-EDTA buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). DNA elution and reverse cross-link was performed adding 200 µl of elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% SDS, 50 µg/ml proteinase K (Sigma-Aldrich, Darmstadt, Germany)) to immune complexes and to input. Both were incubated for 2 h at 68°C, 1300 rpm on a thermomixer. Beads were captured and supernatants were transferred in new 1.5 ml tubes. DNA was isolated by DNA Clean & Concentrator-5 kit (Zymo Research, Orange, CA, USA) according to the manufacturer's protocol. Immunoprecipitated isolated DNA as well as DNA input was analyzed by real-time qPCR.

The primers for PCR amplification were designed with PRIMER 3 [59] (Table S1). Analysis was performed using SensiFAST™ SYBR®Lo-ROX Kit (Bioline Reagents, London, UK) on a DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research). The amount of immunoprecipitated DNA at Cnr1 and Faah promoter regions were normalized to their respective DNA input, then were calculated by the Delta-Delta Ct (ΔΔCt) method and converted to relative expression ratio 2^(−ΔΔCt) for statistical analysis [54].

Statistics

All results were expressed as mean ± standard error of the mean (SEM). Statistical differences between the two experimental groups (CTRL: n = 8; ISO: n = 8) were evaluated using GraphPad Prism® 9 (Graph-Pad Software, San Diego, CA). Nonparametric Mann–Whitney test was used to assess changes in gene expression levels. CpG sites’ DNA methylation level was analyzed using the Mann–Whitney test and multiple comparison with Sidak–Bonferroni correction. All the correlations were performed by Spearman’s rank coefficient. P-values < 0.05 were considered to be statistically significant.

In order to evaluate whether social isolation evokes changes in the regulation of ECS genes expression, we first analyzed the mRNA abundance in the selected brain regions for both CTRL and ISO experimental groups (CTRL and ISO). Overall changes in ECS mRNA levels are reported in Table 1 and Figs. 1 and 3 (in regard to specifically Cnr1 and Faah). Significant alterations have been observed in Cnr1 expression level in the Pfc (Table 1 and Fig. 1) as well as in Cnr1 and Faah mRNA levels in the Amy (Table 1 and Figs. 1 and 3). No changes of any ECS components occur in the Hyp, NAc and Hip (Table 1).

Table 1

Table 1 ECS gene expression levels expressed as 2^(-ΔΔCt) values calculated by Delta–Delta Ct (ΔΔCt) method in distinct brain regions from each animal. Expression was normalized to Gapdh and β-actin. Data are reported as mean ± SEM. Significant differences. Gene expression levels with significant differences between the two groups (p value <0.05, Mann-Whitney test) and relative p-values are reported in bold

Prefrontal cortex (Pfc)
Genes	CTRL	ISO	p values
Genes	Mean ± SEM	Mean ± SEM	p values
*Cnr1*	1.02 ± 0.08	0.65 ± 0.09	0.043
Cnr2	1.05 ± 0.13	0.79 ± 0.12	0.181
Trpv1	1.10 ± 0.60	0.44 ± 0.12	0.413
Faah	1.04 ± 0.11	0.78 ± 0.12	0.142
Magl	1.00 ± 0.04	0.79 ± 0.11	0.236
Daglα	1.03 ± 0.10	1.06 ± 0.18	0.949
Nape-pld	1.05 ± 0.13	0.87 ± 0.13	0.282
Amygdala (Amy)
*Cnr1*	1.08 ± 0.15	0.54 ± 0.11	0.014
Cnr2	1.12 ± 0.20	0.72 ± 0.18	0.228
Trpv1	1.18 ± 0.38	1.02 ± 0.28	0.876
*Faah*	1.04 ± 0.13	0.49 ± 0.12	0.013
Magl	1.08 ± 0.18	1.40 ± 0.32	0.852
Daglα	1.18 ± 0.31	1.04 ± 0.21	> 0.999
Nape-pld	1.04 ± 0.13	1.10 ± 0.32	0.755
Hypothalamus (Hyp)
Cnr1	1.03 ± 0.09	1.26 ± 0.14	0.278
Cnr2	1.12 ± 0.21	1.12 ± 0.23	0.959
Trpv1	1.15 ± 0.21	1.23 ± 0.18	0.878
Faah	1.05 ± 0.11	1.05 ± 0.13	0.981
Magl	1.16 ± 0.25	1.18 ± 0.07	0.382
Daglα	1.06 ± 0.11	1.36 ± 0.14	0.195
Nape-pld	1.03 ± 0.08	0.91 ± 0.09	0.442
Nucleus accumbens (NAc)
Cnr1	1.05 ± 0.13	1.28 ± 0.06	0.165
Cnr2	1.38 ± 0.48	1.34 ± 0.49	0.866
Trpv1	1.06 ± 0.37	0.90 ± 0.24	0.866
Faah	1.24 ± 0.42	0.89 ± 0.18	0.189
Magl	1.35 ± 0.49	1.05 ± 0.30	0.802
Daglα	1.04 ± 0.12	0.87 ± 0.06	0.336
Nape-pld	1.07 ± 0.56	0.80 ± 0.07	0.366
Hippocampus (Hip)
Cnr1	1.01 ± 0.06	1.16 ± 0.07	0.160
Cnr2	1.06 ± 0.14	1.26 ± 0.14	0.228
Trpv1	0.96 ± 0.27	0.51 ± 0.07	0.097
Faah	1.01 ± 0.06	1.10 ± 0.13	0.721
Magl	0.73 ± 0.08	0.98 ± 0.20	0.477
Daglα	1.00 ± 0.03	1.10 ± 0.19	0.672
Nape-pld	1.06 ± 0.14	1.11 ± 0.12	0.593

Cnr1 gene transcription regulation in rats subjected to SI

A significant downregulation of Cnr1 mRNA level in the Pfc of socially isolated (ISO) rats when compared with group-housed controls (CTRL) (ISO: 0.65 ± 0.09; CTRL: 1.02 ± 0.08; p = 0.043) was reported (Fig. 1b). Consistently, we also observed a significant increase in H3K27Me3 levels at gene promoter region in ISO rats when compared with CTRLs (ISO: 2.65 ± 0.51; CTRL: 1.08 ± 0.16; p = 0.006 (Fig. 1c) and these changes are inversely correlated with those of gene expression (Spearman r = − 0.5429; p = 0.0479) (Fig. 1d).

Cnr1 mRNA levels resulted also significantly reduced in the Amy of ISO rats (0.54 ± 0.11) versus CTRL (1.08 ± 0.15) rats, p = 0.014 (Fig. 1e). This data is consistent with a significant decrease in H3K9Ac levels at the promoter of the gene (ISO: 0.71 ± 0.12; CTRL: 1.15 ± 0.06; p = 0.013) (Fig. 1f), positively correlated with the changes in gene expression (Spearman r = 0.8165; p = 0.0033) (Fig. 1g).

No differences in DNA methylation levels between the two groups of rats, considering single CpG sites as well as the average of them in the CpG island analyzed at Cnr1 gene (Fig. 1a) have been observed (Fig. S1a, b).

Interestingly, we also show a significant positive correlation between Cnr1 and Oxtr mRNA levels already reported to be downregulated in the Pfc of ISO rats [36] (Spearman r = 0.7714, p = 0.0019) (Fig. S2).

When considering behavioral data, a significant inverse correlation is observed between Cnr1 gene expression level and the scores obtained in the Hole Board and Open Field tests performed in our previous study [36] in both Pfc and Amy. The reduction in Cnr1 mRNA levels in the Pfc resulted inversely correlated with the increase in the number of head dipping (Spearman r = − 0.8000; p = 0.0016) and in the wall rearing frequency (Spearman r = − 0.6281, p = 0.0243) and, consistently, it can also be observed a positive correlation between the increase in H3K27Me3 levels at Cnr1 promoter and the behavioral outcomes (Spearman r = 0.6571; p = 0.0126) (Spearman r = 0.7084; p = 0.0041) (Fig. 2a, b).

The same correlation profiles were found in the Amy where an inverse correlation is present between Cnr1 mRNA levels with number of head dipping (Spearman r = − 0.5923; p = 0.0358) and rearing frequency (Spearman r = − 0.6556; p = 0.0175), as well as between H3K9Ac levels at Cnr1 gene with Hole Board (Spearman r = − 0.6574; p = 0.0169,) and Open Field (Spearman r = − 0.6676; p = 0.0149) (Fig. 2c, d).

Faah gene transcription regulation in rats subjected to SI

A significant downregulation of Faah mRNA levels in the Amy of ISO rats when compared to CTRL group was observed (ISO: 0.49 ± 0.12; CTRL: 1.04 ± 0.13; p = 0.013) (Fig. 3b).

A consistent significant increase in H3K27Me3 levels was shown at gene promoter region in ISO rats (3.14 ± 0.51) versus CTRLs (1.22 ± 0.26), p = 0.011 (Fig. 3c), and these data significantly inversely correlate with the reduction in Faah expression in this brain region (Spearman r = − 0.6364; p = 0.0299) (Fig. 3d).

No significant alterations in Faah gene DNA methylation levels at single CpG sites as well as in the average of all CpG sites under study in were observed (Fig. 3a and Fig. S1c).

Finally, considering behavioral data, there is also an inverse significant correlation between Faah gene expression levels and data obtained from Hole Board (Spearman r = − 0.7012; p = 0.0066) and Open Field (Spearman r = − 0.6454; p = 0.0148) tests. Behavioral data were found to be also positively correlated with the increase in H3K27Me3 levels at Faah promoter region (Spearman r = 0.7696; p = 0.0019) (Spearman r = 0.6469; p = 0.0145) (Fig. 4a, b).

The first main result of our study is that SI in rats evokes selective alterations in ECS genes expression in the Pfc and Amy, two brain regions with strong connections [60] and which interactions are important in the modulation of affective states and social behaviors [61–64]. Namely, we report significant reductions in mRNA levels for Cnr1 in both brain regions and for Faah gene in the Amy. Several evidence reporting an increased firing at Amy and Pfc might indicate that these events are relevant to evoke the transition from a condition of distress to a possible psychiatric disorder as reviewed by Davidson [65]. Stress exposure firing this specific circuit has been already associated with the downregulation of the ECS and suggested as relevant for the development of an anxious phenotype [66]. No changes were evident in any ECS genes in the other three brain regions here analyzed.

It is well-known that Cb1 receptors mediate most of the effects evoked by cannabinoids in the CNS [67] and we here further support their role in the regulation of stress and emotions, suggested also by their high distribution in the Pfc and Amy, as well as in motivational and cognitive processing [68]. Reduction of Cnr1 expression in Pfc was in fact also reported in an AD mouse model, APP/PS1 mice [69]. These mice did not show at early stages (3 months old) the presence of anxiety, as well as alterations in cognition and social-related behaviors. The regulation of Cnr1 has been thus suggested by the authors as possible biomarker in a presymptomatic stage of cognitive impairment and this might be applied also in our SI rats that could develop cognitive impairment. Moreover, our data on reduced Cnr1 expression in SI fit also with other relevant papers tagging the receptor, for instance: Cb1 blockade decreases social play behavior [70], activation of Cb1 receptors in the Amy can attenuate anxiety responses [71, 72], whereas their antagonism or deletion elicits an anxiogenic response [73]. An earlier study already evaluated the effects of SI on ECS genes expression using immunohistochemistry and, consistent with our findings, Cnr1 resulted down-regulated in the Amy [74]. The authors suggested that lower Cnr1 expression in isolated rats might produce a higher possibility to activate G proteins and thus evoke intracellular second messenger system activation in a more efficient way [74].

As we mentioned in the introduction, we recently reported the central role of Oxtr transcriptional regulation in the same group of rats socially isolated for 5 weeks, and in particular we observed a down-regulation of receptor mRNA levels in the Pfc of ISO rats when compared to controls. This alteration correlated with anxiety-like and stereotyped behaviors [36]. OXTR, which is highly expressed by glutamatergic prefrontal cortical neurons, and Cb1, highly expressed in cortical regions, respectively are known to modulate social recognition [75] and underpin human social–emotional functioning [76].

Interestingly, we here show a significant correlation between the altered expression of OXTR and Cb1 receptors. The Pfc is known to subserve highest-order cognitive abilities and social processing, and it is also the most sensitive brain region to the harmful effects triggered by stress [77]. For this reason, we could hypothesize a possible Endocannabinoid-Oxytocinergic systems interplay in the Pfc of SI rats, partially corroborating an already existing theory of an endocannabinoid signalling mediating oxytocin-driven social reward [37]. Further studies, willing for instance to address oxytocin and endocannabinoid levels correlation in our and other experimental models, are clearly needed to support this hypothesis.

The other relevant finding on ECS genes expression was the reduced levels of Faah mRNA in the Amy of SI rats compared to group-housed animals and this might suggest the enzyme role in processing endocannabinoid signalling on Amy neurons and thus the anxiety processes mediated by this region. It has been in fact already suggested that alterations in Faah activity, evoked by stress or compounds targeting the enzyme [78], would impact Amy functions, including anxiety mechanisms associated with it [42]. It should be considered that ECS genes expression in rat models of SI has been previously investigated by others [74, 79], however beside what already discussed above, other alterations in ECS components’ mRNA have been reported, not consistent with the present findings. Different experimental protocols for SI (8 vs 5 weeks of SI) and methodological approaches (immunohistochemistry protocol vs Real Time PCR) may account for these discrepancies.

Of relevance and consistent with our findings, the down-regulation of Cnr1 [80–83] and reduced levels of FAAH [84, 85] have been also reported in cannabis users, experiencing loneliness [86] and negative effects on social and interpersonal relationships [87].

In order to further analyze the role of epigenetic mechanisms in the downregulation of Faah and Cnr1 genes, we investigated DNA methylation at specific CpG sites and selected histone modifications relevant for activation and inhibition of genes transcription, at the level of both gene promoter regions. We did not report significant differences in DNA methylation levels at any CpG sites under study for both Cnr1 and Faah when comparing SI rats with controls. Instead, we show an increase in H3K27Me3 levels at the Cnr1 promoter region in the Pfc of SI rats consistent with the reduction in gene expression. The same histone modification resulted to be significantly increased at Faah gene promoter in the Amy of SI rats when compared to controls again consistent with the downregulation of enzyme mRNA levels. These data are thus in agreement with the role of trimethylation of Histone-3-Lysine-27 as a modification associated to gene silencing mediating heterochromatin assembly driven by repressive Polycomb complexes [88, 89].

Other studies already reported the dynamic regulation of H3K27 trimethylation evoked by different environmental cues. For instance, chronic stress in mice induces an increase in the epigenetic mark in the hippocampus associated with a reduction in brain-derived neurotrophic factor (Bdnf) [90]. On the other hand, acute stress reduces hippocampal H3K27Me3 [91]. Higher levels of H3K27Me3 were also reported in the orbitofrontal cortex of suicide completers and associated with reduced TrkB gene expression [92].

In the Amy of SI rats we instead did not report any change in H3K27Me3 at Cnr1 gene promoter, whereas we observed a reduction of the activating mark H3K9Ac which role seems not of relevance in the Pfc in our experimental condition. Again, this modulation of the histone modification is consistent and significantly correlated with the reduction in gene expression. In fact, it is known that histone acetylation is associated with gene activation reducing the affinity between DNA and histones [93].

Isolation rearing of rats was already reported to induce a global decrease in H3K9 acetylation [94], whereas in adult mice it was observed an enhancement of the epigenetic mark [95]. Anyhow, SI has been reported to induce alterations in histone acetylation that could contribute to the pathogenesis of major depressive disorder [96].

It should be also taken into account that all the molecular data shown are correlated with the behavioral outcomes we previously reported, and this further support the relevance of these genes regulation in the effects evoked by SI.

Moreover, our data support the existence of an Endocannabinoid-Oxytocinergic systems interplay in the Pfc of ISO rats and suggest a clear role for the transcriptional regulation of selective genes of the ECS (Faah and Cnr1) in the effects evoked by SI in the Pfc and Amy, also pointing out to their epigenetic regulation via specific histone modifications, whereas DNA methylation seems not to be relevant for these genes regulation in our experimental conditions.

In agreement with what recently suggested [97], we provide further evidence on the need for strategies aimed to restore ECS homeostasis in order to counteract the effects of SI and avoid the development of psychiatric conditions, also suggesting the importance of an environmental trigger that could be used to revert those epigenetic mechanisms accounting for the alterations observed. Of particular relevance in the last years, it is the possible role played by nutrition on the prevention of mental disorders [98]. Dietary constituents can in fact impact epigenetic processes to the point that nutritional epigenetics is becoming a new, rapidly expanding research field. [99]. Taking into consideration what we here report, it has been reported that changes in diet methyl donors can evoke alterations in methylation levels of lysine residues of histones 3. For instance, studies showed that H3K27me3 resulted upregulated by a choline supplementation [100] and reduced by a methyl-deficient diet [101]. Thus, it might be helpful to take into account these aspects and assume a proper diet when there might be an environmental trigger like social isolation that might affect mental health.

Author contributions

All authors contributed to the study conception and design.

Conceptualization: Claudio D’Addario; Methodology: Antonio Girella, Claudio D’Addario, Valeria Buzzelli, Viviana Trezza; Formal analysis and investigation: Antonio Girella, Claudio D’Addario, Valeria Buzzelli, Viviana Trezza; Writing - original draft preparation: Antonio Girella, Claudio D’Addario; Funding acquisition: Claudio D’Addario, Enrico Dainese; Resources: Claudio D’Addario, Enrico Dainese, Viviana Trezza; Supervision: Claudio D’Addario, Viviana Trezza, Martina Di Bartolomeo. All authors read and approved the final manuscript.

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Funding

This research was partially funded by the European Union – Next Generation EU. Project Code: ECS00000041; Project CUP: C43C22000380007; Project Title: Innovation, digitalization and sustainability for the diffused economy in Central Italy – VITALITY.

Intellectual property

We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.

Research ethics

We further confirm that any aspect of the work covered in this manuscript that has involved animal models has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript.

Authorship

We confirm that the manuscript has been read and approved by all named authors.

We confirm that the order of authors listed in the manuscript has been approved by all named authors.

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Endocannabinoid system genes regulation in the effects evoked by social isolation in rats

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Abstract

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INTRODUCTION

MATERIALS AND METHODS

Animal model of social isolation

Molecular studies

Gene expression analysis by Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)

DNA methylation analysis

ChIP-qPCR analysis

Statistics

RESULTS

Cnr1 gene transcription regulation in rats subjected to SI

Faah gene transcription regulation in rats subjected to SI

DISCUSSION

Declarations

Conflict of interest

Funding

Intellectual property

Research ethics

Authorship

References

Additional Declarations

Supplementary Files

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Journal Publication

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