Rationale: The endocannabinoidome mediators, N-Oleoylglycine (OlGly) and N-Oleoylalanine (OlAla), have been shown to reduce acute naloxone-precipitated morphine withdrawal affective and somatic responses.Objectives: To determine the role and mechanism of action of OlGly and OlAla in withdrawal responses from chronic exposure to opiates in male Sprague-Dawley rats.Methods: Opiate withdrawal was produced: 1) spontaneously 24 h following chronic exposure to escalating doses of morphine over 14 days (Experiments 1 and 2) and steady-state exposure to heroin by minipumps for 12 days (Experiment 3), 2) by naloxone injection during steady-state heroin exposure (Experiment 4), 3) by naloxone injection during operant heroin self-administration (Experiment 5).Results: In Experiment 1, spontaneous morphine withdrawal produced somatic withdrawal reactions. The behavioral withdrawal reactions were accompanied by suppressed endogenous levels of OlGly in the nucleus accumbens, amygdala, and prefrontal cortex, N-Arachidonylglycerol and OlAla in the amygdala, 2-arachidonoylglycerol in the nucleus accumbens, amygdala and interoceptive insular cortex, and by changes in colonic microbiota composition. In Experiment 2, treatment with OlAla, but not OlGly, reduced spontaneous morphine withdrawal responses. In Experiment 3, OlAla attenuated spontaneous steady-state heroin withdrawal responses at both 5 and 20 mg/kg; OlGly only reduced withdrawal responses at the higher dose of 20 mg/kg. Experiment 4 demonstrated that naloxone-precipitated heroin withdrawal from steady-state exposure to heroin (7 mg/kg/day for 12 days) is accompanied by tissue-specific changes in brain or gut endocannabinoidome mediator, including OlGly and OlAla, levels and colonic microbiota composition, and that OlAla (5 mg/kg) attenuated behavioural withdrawal reactions, while also reversing some of the changes in brain and gut endocannabinoidome and gut microbiota induced by naloxone. Experiment 5 demonstrated that although OlAla (5 mg/kg) did not interfere with operant heroin self-administration on its own, it blocked naloxone-precipitated elevation of heroin self-administration behavior.Conclusion: These results suggest that OlAla and OlGly are two endogenous mediators whose brain concentrations respond to chronic opiate treatment and withdrawal concomitantly with changes in colon microbiota composition, and that OlAla may be more effective than OlGly in suppressing chronic opiate withdrawal responses. N-Oleoylglycine (OlGly) is an endogenous lipoaminoacid produced in the brain following mild trauma and has been shown to reduce the behavioral consequences of this condition  agonist and interacts with the eCB system through its weak inhibition of the eCB-degrading enzyme, fatty acid amide hydrolase (FAAH)  in mice was prevented by pretreatment with a PPARα antagonist -like mediators, which, together with their receptors and metabolic enzymes, constitute the endocannabinoidome (eCBome) , N-Oleoyondition  as well ondition . OlGly aondition  and somaondition . In vitre (FAAH) , but wittagonist , the atttagonist . Howevertagonist . Thus, iN-Arachidonoylethanolamide (anandamide [AEA]), which is a fatty-acid amide eCB with structural similarities to OlGly, was stabilized by via the addition of a methyl group near the amide linkage to form methanandamide. This compound was more stable against FAAH and possessed greater potency as a ligand for the CB1 receptor. Based on this result, we hypothesized that OlGly could be stabilized against amidase degradation in the body by the addition of a methyl group to the glycine’s alpha carbon, sterically hindering enzymatic cleavage at the amide bond. This newly synthesized lipoaminoacid, N-Oleoylalanine (OlAla), was indeed shown to block the establishment of affective symptoms of naloxone-precipitated MWD for a longer duration than OlGly, and to mediate its effects via CB1 and PPARα receptors. Additionally, OlAla was as effective as OlGly in reducing somatic MWD symptoms, including conditioned gaping (a measure of nausea) to a flavor paired with MWD. Consistently, OlAla was found to inhibit FAAH and activate PPARα in vitro  microbiota composition. In Experiment 1, rats were injected with escalating doses of morphine or saline every 12 h for 14 days. Twenty-four hr after the final injection, spontaneous somatic symptoms were measured. Immediately following testing, animals were sacrificed, and brain tissues were taken from the nucleus accumbens (NAc), prefrontal cortex (PFC), interoceptive insular cortex (IIC) and the amygdala. Within these brain regions, levels of the eCBome mediators, AEA and 2-arachidonoylglycerol (2-AG) (two eCBs acting at CB1 and CB2 receptors), oleoylethanolamide (OEA), palmitoylethanolamide (PEA) and other N-acylethanolamines, OlGly, N-arachidonoylglycine (AraGly), and OlAla, among others, were quantified by LC-MS-MS. Colonic samples were also taken at the end of the experiment to look at the possible concomitant changes in gut microbiota composition. Experiment 2 was conducted as Experiment 1, except that the rats were pretreated with Vehicle, OlGly or OlAla 10 min prior to spontaneous or naloxone-precipitated withdrawal. In Experiment 3, rats were implanted with osmotic minipumps containing heroin (because of solubility limits for appropriate doses of morphine via minipumps) for 12 days. On day 12, rats were treated with Vehicle, OlGly  or OlAla  2, 4, and 24 h following removal of the pumps prior to spontaneous somatic withdrawal testing. In Experiment 4, the effect of OlAla on naloxone-precipitated heroin withdrawal was evaluated on Day 12 in rats with intact osmotic minipumps containing saline or heroin (Exp. 4), with brain and gut samples again evaluated for eCBome mediator levels and microbiota composition, in this case allowing to distinguish between the effects on these endpoints of both chronic treatment with, and withdrawal from, chronic opiate administration. Finally Experiment 5 evaluated the potential of OlAla to interfere with operant heroin self-administration on a Fixed Ratio 1 (FR1) schedule and naloxone-precipitated elevation of heroin self-administration.The current study aims to expand on the previous findings on the role of OlGly and OlAla in opiate withdrawal by using male Sprague Dawley rats undergoing spontaneous and naloxone-precipitated opiate withdrawal. We evaluated if OlGly and OlAla: 1) are present in specific brain areas involved in addiction and withdrawal symptoms; 2) reduce behavioral withdrawal responses and do so in a manner mediated by activation of the CBA total of 243 male Sprague Dawley rats were acquired at 200–225 g from Charles River Laboratories  and began experimental testing at an average weight of 300–325 g (average age of 60–70 days). The rats were pair-housed in Plexiglas chambers and located in a colony room maintained on a 12-h reversed light/dark cycle (7:00/19:00) with lights on at 19:00 h, and a temperature of 21 C. Standard rat chow  and water were provided ad-libitum. Following their arrival, rats were given 1 week of acclimation to the testing facility prior to any experimental procedures. All experimental testing began 2 h into the rat’s dark cycle and were approved by The University of Guelph Animal Care Committee and adhere to the guidelines of the Canadian Council on Animal Care.Oleoylglycine (OlGly) and Oleoylalanine (OlAla) were provided by the Mechoulam laboratory, and administered intraperitoneal (i.p.) at a dose of 5 and 20 mg/kg in a volume of 1 ml/kg. Both compounds were dissolved in a glass graduated cylinder in ethanol with Tween 80 added to the solution, the ethanol was then evaporated off with a nitrogen stream, after which saline was added . Vehicle (1 ml/kg) for cannabinoid compounds consisted of a 1:9 mixture of Tween80 to saline. Naloxone (1 mg/ml) was prepared with physiological saline and administered subcutaneously at a dose of 1 mg/kg.In Experiment 1 and 2, morphine  was prepared with physiological saline and administered subcutaneously (s.c) at a volume of 2 ml/kg to result in the respective doses: 10, 20, 30, 40, 50, 60, 70, 80 mg/kg.In Experiments 3 and 4, the osmotic minipumps (Alzet Model 2002) used express 0.5 ul/hour for a period of 14 days . Because animals weighed an average of 300 g over the course of the experiment pumps were filled with a heroin solution (following the Alzet pump instructions) at a concentration of 175 mg/ml to result in a 7 mg/kg dose of heroin daily.In Experiment 5, Heroin (OVC pharmacy) was prepared in a saline vehicle at a concentration of 4 mg/ml. This stock was diluted in the liquid infusion pumps according to daily weights to ensure rats received doses of 0.05 mg/kg/infusion during self-administration.All surgical manipulations occurred following acclimatization to the colony room. In Experiments 3 and 4 rats were surgically implanted with osmotic minipumps under isoflurane anesthesia. Briefly, a small incision (approximately 3 cm) was made on the skin beginning approximately 2 cm caudal to the scapulae of the animal. Using a hemostat to spread apart the subcutaneous layer a pocket was formed in a caudal direction. The pump was then inserted into the pocket with the flow moderator pointing caudally and the incision was closed with a wound clip . The animal was closely monitored following surgery until they displayed normal respiration, posture, and locomotor activity.In Experiment 5, rats were surgically implanted with intravenous catheters under isoflurane anesthesia according to the procedure described by Minhas and Leri (2014). Following catheter implantation rats were monitored for 1 week prior to the first day of operant self-administration.N-acylserotonin quantification by isotope dilution . Then, the lipid extract was dissolved in 100 ul of CH3OH and analyzed by either LC-APCI-MS for AEA, 2-AG, PEA, OEA, DHEA, and EPEA quantification, which were calculated based on their area ratio with the internal deuterated standard signal areas . The chromatograms of the high-resolution [M-H]- values were extracted and used for calibration and quantification. LC analysis was performed in the isocratic mode using a Phenomenex Kintex Polar C18 column  and CH3OH/water/formic acid (85:15:0.1 by vol.) as the mobile phase with a flow rate of 0.15 ml/min. Identification of N-acylglycines, N-acylserotonins and N-oleoylalanine was carried out using ESI ionization in the negative mode with nebulizing gas flow of 1.5 ml/min and curved desolvation line temperature of 250°C. OA5HT was quantified using the peak of deuterated AA5HT as internal standard. The quantification was performed by isotope dilution by using m/z values of 340.2836 and 338.2672 corresponding to the molecular ion [M-H]- for deuterated and undeuterated OlGly, respectively; m/z values of 368.3027 and 360.2830 corresponding to the molecular ion [M-H]- for deuterated and undeuterated AraGly, respectively; m/z values of 356.3159 and 352.2835 corresponding to the molecular ion [M-H]- for deuterated and undeuterated OlAla, respectively; 469.3310 and 461.3319 corresponding to the molecular ion [M-H]- for deuterated and undeuterated AA5HT, respectively; or m/z value of 439.2873 corresponding to the molecular ion [M-H]- of undeuterated OA-5-HT. The most dominant product ion for each lipid class  was selected for MRM.Brain and intestinal tissues were frozen in liquid nitrogen immediately after dissection, which took place within 5 min from sacrifice. Tissues were dounce-homogenized and extracted with chloroform/methanol/Tris-HCl 50 mM pH 7.5  containing internal deuterated standards for AEA, 2-AG, PEA, OEA, AraGly, OlGly, and OlAla and al areas , and by p values, which were all higher than 0.05.At least 10 mediators were measured for each of the analysed six tissues belonging to either 2 or 8 experimental groups, for Experiment 1 and 4, respectively, thus generating at least between 120 and 480 measures, and a huge amount of comparisons to discuss. For this reason, we decided not to show mediator data for which we found no statistically significant differences among groups, nor the corresponding phyloseq and vegan R packages.DNA was extracted from intestinal contents using the QIAmp PowerFecal DNA kit  according to the manufacturer’s instructions. The DNA concentrations of the extracts were measured fluorometrically with the Quant-iT PicoGreen dsDNA Kit  and the DNAs were stored at −20°C until 16S rDNA library preparation. Briefly, 1ng of DNA was used as template and the V3-V4 region of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the QIAseq 16S Region Panel protocol in conjunction with the QIAseq 16S/ITS 384-Index I  kit . The 16S metagenomic libraries were eluted in 30 µl of nuclease-free water and 1 µl was qualified with a Bioanalyser DNA 1000 Chip  to verify the amplicon size (expected size ∼600 bp) and quantified with a Qubit . Libraries were then normalized and pooled to 2 nM, denatured and diluted to a final concentration of 6 pM. Sequencing (2 × 300 bp paired-end) was performed using the MiSeq Reagent Kit V3 (600 cycles) on an Illumina MiSeq System. Sequencing reads were generated in less than 65 h. Image analysis and base calling were carried out directly on the MiSeq. Data was processed using the DADA2 pipeline  and assip values, which were all higher than 0.05.Hundreds of bacterial taxa were measured for each of the analysed 2 intestinal tissues, belonging to 2 or 8 experimental groups, for Experiment 1 and 4, respectively, thus generating hunderds of measures, and an incredible amount of comparisons to discuss. For this reason, we decided not to show taxa for which we found no statistically significant differences among groups, nor the corresponding Somatic withdrawal behaviors were recorded in an observation chamber (22.5 cm × 26.0 cm × 20.0 cm) constructed with four black opaque Plexiglas walls and a lid. This chamber sat on top of a clear glass table with a video camera (Panasonic WV-CP484) placed below to record the ventral surface of the rat. Videos were sent to a computer containing The Observer Software  which was used to score somatic behaviors.In Experiment 5, Plexiglas operant conditioning chambers  were used for self-administration procedures. Each chamber was located within a larger sound-attenuating partition, with a built-in fan to mask noise and provide ventilation. An overhead house light (28 V) was situated on the back wall of each chamber. The front wall contained a retractable lever (8 cm above floor level) which was situated directly below a cue-light  as well as a stationary lever (8-cm above floor level). The retractable, “active”, lever was linked to a liquid infusion pump  that was set to deliver a 150 μl/kg heroin infusion over a 5 s interval following a lever-press. The stationary lever served as the “inactive lever” and produced no consequence when manipulated. Stainless steel bars (30.5 cm × 24.1 cm × 21.0 cm) provided the foundation of the conditioning chambers. A MED-PC interface controlled all conditioning chambers and recorded the number of infusions received, and responses emitted on the active and inactive lever.n = 5) or saline (n = 4) for 14 days. On the first day of chronic injections, rats were given 10 mg/kg morphine (AM/PM). The dose was then progressively increased every 2 days  until receiving one final AM 80 mg/kg dose on Day 14.A total of nine rats were assigned to receive chronic s.c. exposure to escalating doses  of morphine  and gut tissues (colon) were removed, flash frozen and stored at −80°C for subsequent eCBome analysis by LC-MS-MS.n = 8/group) were: Chronic Saline -VEH-Saline, Chronic Saline-OlGly Saline, Chronic Saline-OlAla-Saline, Chronic Saline VEH-Naloxone, Chronic Saline OlGly-Naloxone, Chronic Saline OlAla-Naloxone, Chronic Morphine -VEH-Saline, Chronic Morphine OlGly Saline, Chronic Morphine-OlAla-Saline, Chronic Morphine VEH-Naloxone, Chronic Morphine OlGly-Naloxone, and Chronic Morphine OlAla-Naloxone.A total of 96 rats evaluated the potential of OlGly  and OlAla  to reverse withdrawal reactions 24 h following from chronic morphine or saline exposure. Half of the rats were tested following an injection of saline and half were tested following an injection of naloxone (s.c.). Twenty-four hr following the final chronic saline or morphine injection on Day 15, the rats were injected with VEH, 5 mg/kg OlGly or 5 mg/kg OlAla 10 min prior to an injection of saline or naloxone and 10 min later were placed in the somatic withdrawal chamber for 30 min during which the rats were videotaped and later scored for withdrawal behaviors. The groups (n = 7–8/group) are designated by the pretreatment injection at the time of withdrawal testing and the chronic treatment by minipumps : Group VEH-Saline (n = 8), Group VEH-Heroin (n = 8), Group 5 OlGly-Heroin (n = 7), Group 5 OlAla-Heroin (n = 8), Group 20 OlGly-Heroin (n = 8) and Group 20 OlAla-Heroin (n = 7). Rats received the pretreatment injection  as well 2 and 4 h after removal of the pumps to enhance the clinical relevance by increasing the likelihood of exposure to the treatment with onset of withdrawal.A total of 46 rats were in this experiment, 38 of which were implanted with minipumps containing heroin and eight rats were implanted with minipumps containing saline. The pumps remained intact for 12 days. Twenty-four hr following removal of the pumps, the rats were placed in somatic withdrawal observation chambers (for 30 min) following a pretreatment injection of VEH, OlAla  or OlGly . The groups (n = 28) or heroin (n = 28). On Day 12, with the pumps intact, the rats were injected with VEH or OlAla (5 mg/kg) 10 min prior to an injection of the withdrawal treatment drug, saline or naloxone. Ten min after the final injection the rats were placed in the somatic withdrawal observation chambers for 30 min. Immediately after removal from the somatic withdrawal chambers, the brain and gut tissue was collected and stored at −80°C as in Experiment 1. The groups (n = 7) were: Chronic saline-VEH-saline, Chronic saline-OlAla-saline, Chronic heroin-VEH-saline, Chronic heroin-OlAla-saline, Chronic saline-VEH-naloxone, Chronic saline-OlAla-naloxone, Chronic heroin-VEH-naloxone and Chronic heroin-OlAla-naloxone.A total of 56 rats were implanted with osmotic minipumps filled with saline (n = 8/group) were implanted with heroin minipumps and on Day 12, were given the CB1 antagonist, AM251 , or PPARα antagonist, MK886 , 20 min prior to OlAla  10 min prior to naloxone. The doses of these compounds used have been previously demonstrated to have no effect on their own on naloxone induced opiate somatic withdrawal responses . The rats were then immediately placed in the observation chamber for 30 min and videotaped.To determine the mechanism of action of the effect of OlAla on naloxone-precipitated heroin withdrawal, an additional two groups (n = 9) or OlAla (n = 11) matched for infusions earned and responses (active and inactive) made on the last day of acquisition (session 10). Two baseline sessions were interspersed between each test session to allow self-administration behavior to stabilize following treatment.A total of 20 rats completed the experiment. All self-administration sessions ran daily for 180 min and began 2 h into the rat’s dark cycle. Intravenous catheters were flushed with saline prior to each session to ensure there were no obstructions in the drug delivery line. Once placed in conditioning chambers rats were habituated for 5 min prior to each test session. Illumination of the house light signaled the beginning of a session, with the active lever extending out after a 10 s delay. A continuous schedule was used to reinforce responding on the active lever in which rats received a 150 μl/kg intravenous infusion of heroin (0.05 mg/kg/infusion) and the presentation of a cue-light for 5 s. During this time, additional responses on the active lever were recorded but did not lead to additional reinforcement. Responding on the inactive lever had no programmed consequences and served as a baseline measure of non-reinforced operant responding. The acquisition phase of self-administration lasted for 10 sessions. For the first 3 days, animals were primed (up to 6 primes per session) if no active lever response was emitted within a 15 min period. During sessions 7–10, rats were adapted to the injection procedure with the i.p. administration of 1 ml/kg of VEH 10 min prior to 1 ml/kg s.c saline 5 min prior to being placed in the conditioning chambers. Rats in both groups were maintained on this injection schedule throughout the experiment except during test sessions when the drug group received the appropriate treatment. Following acquisition, rats were assigned to VEH (n = 11) received OlAla  and rats in in the VEH group (n = 9) received VEH 10 min prior to s.c. saline, 5 min prior to being placed in the conditioning chambers to test whether OlAla itself modified heroin self-administration.On Session 11 all rats in the drug group  10 min prior to naloxone  5 min prior to placement in the conditioning chambers.ex vivo levels of OlGly, AraGly, AEA, 2-AG, OEA, and PEA were analyzed by independent t tests for the chronic saline and chronic morphine group. In Experiment 2, the effect of the pretreatment on the withdrawal behavioral responses were analyzed a 2 (chronic treatment) × 3 (pretreatment) × 2 (test treatment) between groups analysis of variance (ANOVA). In Experiment 3, each of the somatic withdrawal behaviors scored was entered into a one-way ANOVA for groups with subsequent comparisons by Bonferroni post hoc tests. In Experiment 4, the number or duration of each of the behavioral measures were entered into a 2 × 2 × 2 factorial design, with the between group factors of chronic treatment , withdrawal treatment  and pretreatment (VEH or OlAla). To determine the mechanism of action of the interference with naloxone-precipitated heroin withdrawal, the relevant behavioral withdrawal measures for each of the additional two groups (MK886-OlAla and AM251-OlAla) was compared with group heroin-naloxone-OlAla in a single factor ANOVA. Variations in brain biochemicals and fecal microbiota compositions were evaluated using one-way analysis of variance (ANOVA) and Tukey HSD post-hoc tests to assess treatment effects (VEH vs drug groups). For Experiment 5, the self-administration acquisition period, sessions 1–10, the number of infusions received were entered into a repeated measures ANOVA. The number of responses made on the active lever and the inactive lever were entered into a 2 × 10 repeated measures ANOVA with lever (active or inactive) and session (sessions 1–10) as within subject factors. To evaluate the effect of OlAla alone on heroin self-administration on Day 11, an independent t-test was conducted between the VEH and drug group for each self-administration behavior. The VEH-Naloxone and OlAla-Naloxone data for each measure on Days 14 and 17 were pooled, because the order of testing did not differ for any measure as assessed by paired t-tests. The self-administration behaviors for Group VEH, were each entered into a paired t-test across the two test days , which revealed no significant differences; therefore, the data for this group was pooled across these days for comparison with the drug group on each test. For the drug group each test measure was then entered into a paired t-test . Finally, each measure for the VEH group was compared with the drug group on all tests. Statistical significance was defined as p < 0.05. Box plots borders represent the first and third quarter while the intersecting line represents the median of data. Whiskers representing 1.5× the interquartile range. Samples falling beyond this range are drawn as dots.In Experiment 1, the somatic withdrawal behaviors and the Rats displayed somatic withdrawal responses 24 h following chronic escalating doses of morphine. These behaviors were accompanied by a suppression of endogenous OlGly in the NAc, amygdala and PFC, as well as suppressed AraGly in the amygdala and suppressed 2-AG in the NAc, amygdala, and IIC.p = 0.034, enhanced wet dog shakes, t(7) = 3.1; p = 0.016, elevated mouthing movements, t(7) = 2.4; p < 0.05, and suppressed active locomotion, t(7) = 2.4; p = 0.046, relative to rats in the chronic saline group. The groups did not differ in instances of diarrhea.p = 0.028, amygdala, t(7) = 3.0; p = 0.02, and PFC, t(7) = 3.9; p = 0.006, but not in the IIC. Morphine treated rats also showed significantly reduced AraGly in the amygdala, t(7) = 2.6; p = 0.037 and reduced 2-AG in the NAc, t(7) = 2.4; p = 0.048, amygdala, t(7) = 2.9; p = 0.0379, and the IIC, t(7) = 29.0; p < 0.001. Chronic morphine followed by a 24 h withdrawal period did not modify levels of AEA, OEA or PEA in any region.p < 0.001).We also measured the levels of endogenous brain OlAla, which was detected only in the amygdala. Interestingly, OlAla was significantly suppressed in this brain area  of rats undergoing chronic morphine administration followed by a 24 h withdrawal period, t(7) = 5.6; Firmicutes:Bacteroides ratio  = 6.5; p = 0.003. Rats treated with OlAla displayed fewer mouth movements than rats treated with VEH (p < 0.01) and OlGly (p = 0.04) by Bonferroni tests.OlAla, but not OlGly, blocked mouthing movements, and attenuated the locomotor deficits produced by withdrawal from chronic morphine, when pooled across naloxone and saline test groups. The behaviors of diahrrea and abdominal contractions did not reveal significant effects of pretreatment. p < 0.001, test treatment, F = 23.3; p < 0.001, chronic exposure x pretreatment, F = 3.5; p = 0.036 and chronic exposure by post-treatment, F = 7.2; p = 0.009. Chronic morphine produced suppressed locomotion relative to chronic saline and rats tested with naloxone produced suppressed locomotion relative to saline. Among the chronic saline groups, the pretreatment effect was not significant, but among the chronic morphine groups the pretreatment effect was significant, F = 9.1; p < 0.001. Pooled across test drugs, following withdrawal from chronic morphine, rats treated with OlAla were significantly more active than those treated with VEH (p < 0.01) or OlGly (p < 0.01) by Bonferroni tests. The 2 × 3 × 2 ANOVAs for the additional behaviors (not depicted) also revealed significant effects of chronic treatment for abdominal contractions, F = 40.1; p < 0.001) and diarrhea, F  = 0.001, but neither behavior interacted with the pretreatment variable.For active locomotion, the 2 × 3 × 2 factorial ANOVA revealed a significant effect of chronic exposure, F = 99.9; The only withdrawal behaviors that revealed significant group differences were those of mouthing movements and abdominal contractions. The withdrawal responses of abdominal contractions were suppressed in Groups 5 OlAla-heroin, 20 OlAla-heroin and 20 OlGly-heroin. The withdrawal response of mouthing movements was only suppressed in Group 5 OlAla-heroin.p = 0.05 and abdominal contractions, F(5.40) = 9.2; p < 0.001. Subsequent Bonferroni post hoc comparison tests revealed that Group V-heroin displayed significantly more mouthing movements (p < 0.01), abdominal contractions (p < 0.001) than Group V-saline. As well, Group 5 OlAla-heroin, Group 20 OlAla-heroin displayed fewer (p’s < 0.01) abdominal contractions than Group V-heroin. Group 5 OlAla-heroin also displayed fewer mouthing movements (p < 0.05) than Group V-heroin. There was no difference among the groups in duration of active locomotion F = 0.86; p = 0.52.p = 0.002, withdrawal treatment, F = 80.9; p < 0.001, a chronic treatment by withdrawal treatment interaction, F = 22.4; p < 0.001, and a chronic treatment by withdrawal treatment by pretreatment interaction, F 11.9; p < 0.001. Subsequent analysis of the withdrawal treatment groups separately revealed no significant effects among the saline treated groups, but among the naloxone treated groups there were significant effects of chronic treatment, F = 19.1; p < 0.001, and a withdrawal treatment by chronic treatment interaction, F = 9.7; p = 0.005. Among the naloxone treated groups, only in the chronic heroin treated rats pretreated with VEH displayed elevated abdominal contractions, OlAla interfered with elevated abdominal contractions (p < 0.025).OlAla (5 mg/kg) reduced the naloxone-precipitated withdrawal responses of abdominal contractions and mouth movements, as can be seen in p < 0.001, withdrawal treatment, F, 49.3; p < 0.001, pretreatment, F = 10.3; p = 0.003, a chronic treatment by withdrawal treatment interaction, F = 45.6; p < 0.001, a chronic treatment by pretreatment interaction, F = 7.9; p = 0.007, a withdrawal treatment by pretreatment interaction, F = 4.1; p = 0.048, and a chronic treatment x withdrawal treatment by pretreatment interaction, F = 4.5; p = 0.031. Subsequent analysis of the withdrawal treatment groups separately revealed no significant effects among the saline treated groups, but among the naloxone treated group there were significant effects of chronic treatment, F = 54.37; p < 0.001, pretreatment, F = 8.2; p = 0.008, and a chronic treatment by pretreatment interaction, F = 7.4; p = 0.012. Among the naloxone treated groups, those chronically treated with heroin showed more mouth movement when pretreated with VEH than when pretreated with OlAla (p < 0.025).The 2 × 2 × 2 factorial ANOVA for the response of mouth movements revealed significant effects of chronic treatment, F = 43.4; p < 0.001 and a chronic treatment by withdrawal treatment interaction, F = 9.45; p = 0.003. Rats chronically treated with heroin displayed suppressed locomotion relative to those chronically treated with saline despite pretreatment conditions; OlAla did not modify this naloxone-precipitated withdrawal reaction. There were no other naloxone precipitated withdrawal effects observed with any of the remaining behavioral measures.The 2 × 2 × 2 factorial ANOVA for the duration of active locomotion revealed only a significant effect of withdrawal treatment, F = 16.3; p = 0.018, and mouth movements, F = 13.4; p < 0.001); subsequent Bonferroni comparison tests revealed that the group pretreated with OlAla alone displayed fewer abdominal contractions (7.2 ± 1.3) and mouth movements (41.6 ± 4.2) than the groups pretreated with either AM251 . We have previously shown that neither antagonist modifies these withdrawal -induced behaviors on their own at these doses . A one way ANOVA among OlAla pretreatment groups  that were treated with naloxone following chronic heroin revealed a significant effect for both abdominal contractions, F = 5.0; se doses .p < 0.05). In the PFC, under conditions of naloxone-precipitated heroin withdrawal, 2-AG levels instead increased, and this effect was not observed following administration of OlAla . Other changes in this area did not appear to be strictly related to heroin treatment or naloxone-induced withdrawal per se, as heroin reduced AEA levels only in the presence of naloxone, without , or with  concomitant OlAla administration (p < 0.001). No significant alterations were observed in the NAc for any of the mediators (data not shown). Perhaps, the most interesting changes were observed in the gut. Particularly, in the jejunum, chronic heroin infusion reduced N-arachidonoylserotonin levels (p < 0.05) with no subsequent effect of naloxone with or without OlAla (p < 0.05), which was not observed in the presence of OlAla (p < 0.05) elevation of both AEA and EPEA to levels similar to those observed in rats only treated with OlAla (N-acylethanolamines (possibly via FAAH inhibition).The effect of chronic heroin and naloxone-precipitated withdrawal therefrom, in the presence or absence of OlAla, on several eCBome mediators  in various brain areas and intestinal sections are shown in stration . Likewisut OlAla , whereasof OlAla . The colof OlAla . Like inof OlAla . AA5HT aof OlAla . Finallyth OlAla , indicatp = 0.009; p = 0.002; p = 0.03, OlAla-VEH vs. OlAla-heroin; p = 0.02). Despite these observed differences, as with morphine, there were no difference in the Shannon alpha diversity indexes between the groups (data not shown), while the Firmicutes: Bacteroides ratio was only increased by heroin within the naloxone group , which contrasts with the effect observed above with morphine (data not shown). Bacterial family relative abundance changes in response to chronic heroin were different between saline and naloxone test groups  without affecting Prevotellaceae. Our analysis identified no changes in the abundance of genera belonging to Clostridiales_vadinBB60, but the genera Prevotellaceae_UCG-001 and Prevotellaceae_Ga6A1 were both increased by heroin , with OlAla rendering the increase insignificant compared to the control only for the latter.Neither acute naloxone nor OlAla  had any effect on the colonic microbiota in the absence of heroin administration (data not shown). Heroin appeared to significantly modify the gut microbiota of rats as determined by PCoA analysis followed by PERMANOVA  and Ruminococcaceae_NK4A214 being decreased and Romboutsia being increased . The analysis of the number of responses made on the active and inactive lever revealed a main effect of session F = 8.6, p < 0.001, lever F = 52.2, p < 0.001 and a significant session × lever interaction F = 10.7, p < 0.001, with the rats demonstrating an increase in active lever, but not inactive lever responses across sessions (data not depicted).The number of infusions received and active lever responses emitted increased across sessions, while the number of inactive lever responses remained consistently low during self-administration acquisition. The analysis revealed a significant effect of Day on infusion received, F = 16.9, Oleoyl alanine alone did not interfere with self-administration of heroin assessed by any measure. p < 0.01), first 30 min infusions (p < 0.01), total active responses (p < 0.025) and first 30 min active responses (p < 0.025) than on the OlAla-Naloxone test; however, they did not significantly differ in total inactive responses or first 30 min inactive responses. As well, on the VEH-Naloxone test, but not on the OlAla-Naloxone test, naloxone enhanced total infusions (p < 0.01), first 30 min infusions (p < 0.01), total active responses (p < 0.025) and first 30 min active responses (p < 0.025) relative to group VEH.Naloxone elevated both the number of infusions and active responses for heroin, effects that were suppressed by pretreatment with OlAla in the pooled tests on Days 14 and 17. 1 and PPARα receptors and accompanied by changes in gut or brain eCBome mediators and gut microbiota composition. These findings implicate endogenous eCBome mediators and the gut microbiota in the behavioral and neurochemical responses produced by exposure to chronic opiates and withdrawal therefrom, and suggest that administration of OlAla and OlGly in vivo attenuates the adverse somatic reactions of opiate withdrawal in rat models.We have provided here data suggesting for the first time that OlGly and/or OlAla, two endogenous lipid mediators belonging to the family of lipoaminoacids of the expanded endocannabinoid system, or eCBome : 1) are N-acylethanolamines, another eCBome family of mediators. In Experiment 2, pretreatment with 5 mg/kg OlAla, but not 5 mg/kg OlGly, reduced spontaneous somatic withdrawal behaviors  produced by chronic escalating doses of morphine. Taken together, results from Experiments 1 and 2 demonstrate that OlAla is an endogenous compound that can be measured in the amygdala of rats, where its concentrations can be decreased by exposure to morphine and/or withdrawal therefrom, and that systemic administration of this compound attenuates spontaneous withdrawal signs in morphine-dependent rats. The amygdala is a neural substrate involved in opiate addiction  reduced abdominal contractions, but only low dose OlAla (5 mg/kg) reduced the abdominal contractions and mouthing movements induced by withdrawal. These dose-dependent effects of OlAla are consistent with the known biphasic effects of eCBome mediators on behaviour , whereas levels of the endocannabinoids, 2-AG and AEA, and the other endogenous PPARα ligands, OEA, and PEA, were not affected in any of the brain regions evaluated. Therefore, the changes in endogenous levels of OlGly in the NAc following acute administration of morphine and naloxone-precipitated withdrawal therefrom are opposite to those occurring following chronically administered morphine and spontaneous withdrawal therefrom. On the other hand, OlGly suppressed withdrawal responses from acute morphine exposure   ), OlAla e (FAAH) . The rede (FAAH) , we leanN-acyl-serotonins in the gut. In view of the activity of: 1) 2-AG as an endogenous agonist of PFC CB1 receptors potentially involved in anxiogenic responses  AraGesponses , and 3) nd TRPV1 , two tarnd TRPV1 , we specnd TRPV1 , and thiN-acyl-serotonin levels in intestinal tissues following chronic heroin administration is similar to that observed in mice following 1 week treatment with a cocktail of antibiotics , who used samples from the mouse cecum, also did not detect any Enterococcus taxa.Interestingly, the reduction of ibiotics , and henibiotics . Conversibiotics , which aibiotics . Howeverecreased , and 3) ecreased , most nod pellet , whereaste users . Thus, oal signs , thus supendence , while fpendence , thus in obesity , possiblN-acylethanolamine, N-acyl-glycine, N-acyl-alanine, and N-acyl-serotonin classes of lipids) in either the brain or intestine or both; 2) accompanied by changes in intestinal microbiota composition; and 3) counteracted by exogenous administration of the two most abundant endogenous members of the N-acyl-glycine and N-acyl-alanine lipoaminoacid families in the brain, i.e., OlGly and OlAla. Our findings suggest that OlGly and, more efficaciously, OlAla are capable of reducing somatic signs of opiate withdrawal, that OlAla also counteracts naloxone-precipitated enhancement of heroin self-administration, and that chronic opiate administration and withdrawal therefrom are accompanied by changes in brain and gut eCBome mediator, and gut microbiota, profiles that, in the case of heroin, are also partly reversed by OlAla. Therefore, our study confirms the hypothesis that OlGly and OlAla are endogenous modulators of pathological alterations induced by substances of abuse, and opiates in particular, potentially exploitable as pharmacological treatments for opiate withdrawal syndrome, and capable of exerting their effects by modulating eCBome signaling at either PPARα or CB1 receptors, or both, possibly also via the increasingly emerging interactions between the eCBome and gut microbiome  accompanied by changes in endocannabinoids and eCBome mediators (belonging to the crobiome . These fthdrawal , while a


Understanding seizure development requires an integrated knowledge of different scales of organization of epileptic networks. We developed a model of “epilepsy-in-a-dish” based on dissociated primary neuronal cells from neonatal rat hippocampus. We demonstrate how a single application of glutamate stimulated neurons to generate spontaneous synchronous spiking activity with further progression into spontaneous seizure-like events after a distinct latency period. By computational analysis, we compared the observed neuronal activity in vitro with intracranial electroencephalography (EEG) data recorded from epilepsy patients and identified strong similarities, including a related sequence of events with defined onset, progression, and termination. Next, a link between the neurophysiological changes with network composition and cellular structure down to molecular changes was established. Temporal development of epileptiform network activity correlated with increased neurite outgrowth and altered branching, increased ratio of glutamatergic over GABAergic synapses, and loss of calbindin-positive interneurons, as well as genome-wide alterations in DNA methylation. Differentially methylated genes were engaged in various cellular activities related to cellular structure, intracellular signaling, and regulation of gene expression. Our data provide evidence that a single short-term excess of glutamate is sufficient to induce a cascade of events covering different scales from molecule- to network-level, all of which jointly contribute to seizure development. Epilepsy is not a single disease, but highly heterogeneous with diverse clinical syndromes, associated etiologies , and many different mechanisms contributing to the epileptic phenotype and known frequent comorbidities. Temporal lobe epilepsy (TLE) is the most common epilepsy syndrome in adults. Hippocampal sclerosis (HS) is a frequent structural brain lesion identified in patients with drug-resistant focal epilepsy submitted to surgical treatment . It is hWhile seizures are commonly defined as the clinical manifestation of an abnormal, excessive, hypersynchronous discharge of a population of cortical neurons , it remaWe recently developed a simple and powerful cell culture model of epileptogenesis using a single transient glutamate-induced neuronal mass discharge to transform a primary dissociated cell culture from neonatal rat hippocampus into a repetitively firing synchronous seizure-like network, i.e., “epilepsy-in-a-dish” . To undeAnimal and tissue preparation was performed as previously described . In shor2 onto an uncoated flask to allow settlement of glial cells and their adhesion to the flask. Remaining cells from the supernatant were harvested at 800 rpm for 8 min at room temperature. Cell pellets were resuspended and cultured in serum-free Neurobasal-A medium supplemented with 2% B27, 0.5 mM GlutaMAX, and 1% penicillin–streptomycin . Cells were plated on poly-D-lysine coated coverslips  at a density of 2.5 × 105 cells/well. Cells were maintained at 37 °C in a fully humidified incubator with 5% CO2. After 24 h, 6 µM Cytosine β-D-arabinofuranoside hydrochloride  was added to inhibit proliferation of remaining glial cells. Neurons were continuously maintained in dispersed culture with their original media supplemented with AraC. After 12 days in vitro, the original culture medium was transiently replaced by physiological treatment solution (10 mM HEPES (pH 7.4), 145 mM NaCl, 10 mM glucose, 1 mM MgCl2, 2.5 M KCl, 2 mM CaCl2, 2 µM glycine) with or without 10 µM glutamate [2 until further experiments.Primary neuronal cell cultures from rat neonatal hippocampus were prepared as described before . Brieflylutamate ,45. CultCellular viability was determined 7 and 15 days after glutamate treatment with propidium iodide (PI) and fluorescein diacetate (FDA). Cells were incubated with 500 nM PI and 160 nM FDA for 10 min in Neurobasal A medium at 37 °C. Semiquantitative measurements of PI/FDA-stained cells were performed using a microcomputer imaging system  equipped to an Olympus XI70 microscope. PI- and FDA-positive cell bodies were tagged on the computer screen and manually counted in four independent visual fields at 20× objective magnification. Cell viability was routinely determined in glutamate-treated cultures and time-matched sham controls.Immunofluorescence staining for Sholl analysis was performed 1, 3, 5, 7, and 15 days after glutamate or sham treatment, for synapse quantification after 3 and 7, as well as for interneuron detection 7 days following initial treatment. Neurons cultured on coverslips were fixed in 4% paraformaldehyde and blocked in tris-buffered saline ) with 1% bovine serum albumin , 2% fish skin gelatin  and Triton X-100  for 2 h at room temperature. Primary antibodies were diluted in blocking solution and applied overnight at 4 °C. Secondary fluorophore-conjugated antibodies were diluted in blocking solution and applied for 4 h at room temperature. Nuclei were dyed with 4′,6-diamidino-2-phenylindole  diluted in blocking solution. For Sholl analysis, primary chicken anti-Map2  and mouse anti-Gfap  and secondary Alexa-647 donkey anti-mouse  and Alexa-488 goat anti-chicken  were used. For interneuron quantification, primary rabbit anti-Calbindin  and secondary Alexa-555 goat anti-rabbit  were used. For synapse quantification, primary rabbit anti-Vglut1 , chicken anti-Vgat , and mouse anti-Bassoon , together with secondary Alexa-488 goat anti-chicken and Alexa-555 goat anti-rabbit , as well as Alexa-647 donkey anti-mouse  were used.Immunofluorescence microscopy for Sholl analysis was performed with the Zeiss MN Observer microscope  and ZEN 2012 SP5 software. Singular neurons were tracked in the periphery of cultures, avoiding overlay of different cells and false-positive attribution of dendrites. All images were calibrated to scale for Sholl analysis afterwards. After processing of images, we used the Sholl analysis plug-in v3.7.0  for Fiji2. All images were acquired under standardized conditions. For synapse quantification, five non-overlapping images per coverslip and six independent preparations per experimental condition were used. Co-localized presynaptic (Bassoon-positive) and postsynaptic (Vglut1- or Vgat-positive) puncta within a mask were counted manually using the cell counter plug-in for Fiji (1.51s “Multi-point” tool provided by Fiji). In a second approach, we used the automated Fiji plug-in Synapse Counter. During quantification, settings for synapse counting were uniformly chosen for all images within an experiment. Results of automatic puncta quantification were always validated by visual inspection and manual counting for a subset of images. All coverslips were coded and processed in a blinded manner. We normalized all puncta counts to the median number of puncta detected on control neurons for a particular coverslip to combine data acquired from different coverslips in different experiments. An average of 50,000 synapses was analyzed per experimental condition and time point.Synapses were visualized using a Zeiss Observer Z1 LSM 780 microscope  equipped with 405/488/555/647 HXP 120 V Fluorescence lamp, 405/488/555/647 LASOS Argon Laser, and 594 Zeiss In Tune Laser, using ZEN 2012 SP5 software. Images were taken with a 1 mm pinhole using a 63× objective covering an area of 134 µm2, 2.5 mM MgCl2, 10 mM Glucose, 10 mM HEPES, pH 7.4, 320 mOsm; [2 was detected with a Plan Apo 10×, 0.45 NA objective, and perfect focus system in every experiment. Fluo-8 AM was excited by Nikon Intensilight C-HGFI Lamp  through a 455–485 nm excitation and a 495 nm dichroic long-pass mirror. Emitted light was passed through a 488 nm filter Cube GFP . Detection was performed with a cooled EM-CCD camera . The culture was recorded with the NIS Elements  software in two different settings: no binning and 200 ms exposure time or 2 × 2 binning and 50 ms exposure time. For all experimental conditions, 5–10 biological replicates from at least three different preparations were analyzed.Calcium imaging was performed 1, 3, 5, 7, 9, and 15 days after glutamate or sham treatment. Cultures were incubated for 30 min at 37 °C with 5 µM Fluo-8 AM  in 1 µg/µL DMSO. The osmolarity of modified Cohen’s Recording Solution  was adjCellular calcium signals were quantified as the average light intensity of manually selected regions of interest  covering cell bodies. Per-pixel standard deviation was used to identify neurons (ROI) with significant changes in calcium signal. Signals from 80–150 neurons were analyzed in every recording session. Individual traces were exported and analyzed in a custom-developed software designed by V.G. in LabView  .p < 0.001) that mapped to enhancers, promotors , or gene bodies were used to identify differentially methylated genes (DMGs). The biological function of gene networks was analyzed using Qiagen Ingenuity Pathway Analysis (IPA) software .WGBS paid service was performed at the Core Unit NGS  and included DNA extraction, bisulfite conversion, library preparation, sequencing, and data analysis. Sequencing was performed with Illumina HiSeq2500 . Raw paired-end FASTQ files  were trimmed with Trim Galore v0.6.2 using cutadapt v2.3, autodetecting and removing Illumina adapter sequences. Trimmed FASTQ files were then merged with zcat/pigz into a single FASTQ file pair per sample. For bisulfite-sensitive alignment and differential methylation calling, Bismark v0.22.1 with hisat2 v2.1.0 mapping was used  from thep < 0.05, and error bars and specified values represent mean and standard error of the mean (SEM), if not stated otherwise.Statistical analysis was performed using GraphPad Prism7 . All points shown in this work represent an average from at least three independent measurements in three independent preparations, including epileptogenic and sham control cultures obtained from the same preparations . Depending on sample size and distribution, paired or unpaired two-tailed t-test (2 groups), ANOVA (≥3 groups), or respective non-parametric tests and corrections for multiple comparisons were performed. Details are provided in the respective Results section. In all figures, asterisks indicate a level of significance of This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.1st week = 49 ± 10 s, n = 18, SLE duration2nd week = 111 ± 6 s, n = 11, p = 0.0014; We previously developed an in vitro model of epileptogenesis based on a self-organized network of interconnected neurons and a few glial cells . StartinComputational analysis and visual comparison of in vitro SLEs with human intracranial stereo-EEG recordings of seizures identified strong similarities and a related sequence of events with defined onset, progression, and termination d. SLEs tCtrl1d = 241.8 ± 70.2 µm, lengthGlu1d = 263.1 ± 76.9 µm, p = 0.33; lengthCtrl3d = 185.9 ± 59.8 µm, lengthGlu3d = 241.1 ± 58.7 µm, p = 0.0003; lengthCtrl5d = 187.6 ± 54.9 µm, lengthGlu5d = 241.1 ± 68.9 µm, p = 0.0014; lengthCtrl7d = 227.4 ± 37.2 µm, lengthGlu7d = 303.8 ± 74.0 µm, p < 0.0001; lengthCtrl15d = 272.9 ± 68.5 µm, lengthGlu15d = 289.5 ± 101.7 µm, p = 0.62).We next thought to analyze whether the functional differences between glutamate-treated and control cultures resulted from structural changes of cultured cells or altered cellular assembly of the networks under study. Therefore, we monitored cultured neurons (Map2 immunoreactivity) for up to 15 days after glutamate treatment and quantified neurite length and sprouting using Sholl analysis . With tit-test, mean branchingCtrl1d = 7.0 ± 4.3, Glu1d = 4.9 ± 3.3, p < 0.0001; Ctrl3d = 4.6 ± 2.8, Glu3d = 3.5 ± 2.3, p < 0.0001; Ctrl5d = 4.8 ± 2.9, Glu5d = 5.2 ± 3.6, p = 0.62; Ctrl7d = 7.8 ± 4.7, Glu7d = 5.9 ± 3.8, p < 0.0001; Ctrl15d = 5.9 ± 4.1, Glu15d = 4.0 ± 3.0, p < 0.0001; When we plotted the number of neurite intersections against distance from soma: neurons following glutamate treatment displayed fewer intersections close to the soma, but more intersections with increasing distance from the soma, as compared with time-matched sham-treated controls . This difference was not yet detectable three days after epileptogenesis induction, i.e., following glutamate exposure . Only synapses co-labeled for Bassoon , a scaffolding protein involved in organizing the presynaptic cytoskeleton of active axon terminals, and Vgat or Vglut1 were included in the measurements  over excitatory synapses stained positive for vesicular glutamate transporter 1 (Vglut1) seven days after glutamate treatment compared with time-matched sham controls , glial fibrillary acidic protein (Gfap), and the interneuronal marker protein Calbindin . A general decline in Calb1-positive cells relative to overall cell numbers was observed in cultures seven days after glutamate treatment  were called, which uniquely mapped to 130 differentially methylated genes (DMGs). We then performed functional pathway analysis using Qiagen’s ingenuity pathway analysis software. There was an enrichment of DMG in the axonal guidance signaling pathway in line with the dominant structural changes that we detected in neuronal networks following glutamate treatment. Next, functional interaction networks of proteins encoded by hyper- (green) and hypomethylated genes (red) were identified and mapped to the cell as a structural and functional unit. Thereby the spatial location of protein nodes was shifted into their corresponding subcellular locations  were identified with an FDR < 0.05 a. Princiocations c. DMGs aocations . Many ofocations . Our datEpileptogenesis, i.e., the process that leads to the development of spontaneous recurrent seizures and disease progression, cannot be studied in the human brain in situ. Only in a subset of patients undergoing epilepsy surgery for treatment of their focal drug-resistant epilepsy do we have access to human brain tissue. However, the described molecular, structural, and functional changes in such tissue are representative of a late (chronic) disease stage and do not allow conclusions to be drawn on when these changes occurred. Therefore, experimental models are needed to study the disease course. Here, we describe a simple but powerful model providing insights into the full process of epileptogenesis in an exceptionally short time frame covering all steps from initial precipitating injury to silent latency period, development of spontaneous recurrent seizure-like events (SLEs), and disease progression. We performed an integrated study addressing multiple scales from molecules to cellular and network structure and neurophysiological function. Our data show striking and broad similarities between our cellular model system with human epilepsy across scales including similar seizure pattern and duration, cell structure, and a specific phenotype-associated gene-regulatory epigenetic profile.Rodent models of chronic epilepsy have been used extensively in the past to study epileptogenesis. Still, they have been proven to be labor- and cost-intensive and challenging to study in sufficient temporal, cellular, and molecular resolution. Acute or organotypic slice cultures are often used for more detailed studies on seizure mechanisms, as they sustain much of the necessary circuitry. While acute preparations are viable for a few hours and valuable for studying acute provoked seizures, organotypic slice cultures are sustainable for multiple weeks. They can be used to study temporal processes, including circuit development, synaptic plasticity, and axonal sprouting. However, interpretation of molecular data from brain tissue with its high cellular heterogeneity remains challenging, as results cannot easily be attributed to a specific cell type. Approaches that help address this issue include cell sorting before molecular analysis, microfluidics and subsequent single-cell sequencing, or culturing select cell populations, all of which have specific technical and biological advances and limitations. Here, we used primary neuronal cells from rat HC that, after maturation in vitro, self-assembled into 3D functional networks  and developed spontaneous recurrent epileptiform activity following a single transient stimulus with physiological levels of glutamate. With a stable 92–96% neuronal phenotype, our cultures could be used for a nearly unbiased study of neuronal features of seizure generation.Epileptogenesis was monitored following glutamate exposure using live-cell calcium imaging at different time points. After 24 h, no specific neuronal activity was detected except for episodic random unsynchronized spikes. At 3 days after the transient glutamate exposure, synchronous spiking activity was detected and remained prominent at 5, 7, 9, and 15 days. SLEs emerged 5 days after treatment and were maintained in the 7-, 9-, and 15-day cultures. Cell type attribution of detected signals was always confirmed by Map2/Gfap immunofluorescence of all calcium-imaged cultures. We identified specific but extremely rare events of glial calcium activity , which only affected individual cells and never occurred in temporal relation with synchronized spiking or SLE development across the network. SLEs in our cultures showed strong similarities with human intracranial EEG seizures, both in terms of duration, stability for around 1–2 min, and according to seizure pattern. The similarities between experimental and human seizure power spectra were striking despite the differences in resolution. Our results indicate the development of spontaneous recurrent epileptiform activity after a short silent period and progressive worsening with more prolonged SLEs over time, thus recapitulating the presumed general pathogenic time course of epileptogenesis. In our study design, no continuous measurement of epileptiform activity was possible. Preparations of cultures on (high-density) multi-electrode arrays would allow measurements of field potentials to examine network activity at higher temporal resolution, however not at single-cell resolution. Other complementary studies could involve whole-cell patch-clamp approaches, enabling manipulations of individual cells and determining downstream small network effects if several cells over the network are monitored simultaneously.Structural changes of neuronal cells during epileptogenesis were studied using Map2 immunofluorescence staining and Sholl analysis. The neurite length of neurons in glutamate-treated cultures was significantly increased early on in the epileptogenic process. This notable change in cellular structure persisted over time, together with a higher degree of terminal branching. Altered cell morphology in our model matched structural changes previously described ex vivo in humans  and ratsKcnh5), an outward-rectifying, non-inactivating potassium channel that regulates neurotransmitter and hormone release. Diseases associated with KCNH5 mutations include early infantile epileptic encephalopathy (EIEE). We also found hypermethylation of collagen type XIV, alpha 1 (Col14a1), an ECM component that is highly expressed in the brain and has been linked to familial adult myoclonic epilepsy (FAME). We further identified hypermethylation of doublecortin (Dcx). Dcx is a microtubule-associated protein involved in neuronal migration. Mutations in human DCX have been identified as being causative in double cortex syndrome and other cortical malformations associated with epilepsy. Hence, although we lack a transcriptomic profile of our cultures, these data support a gene regulatory role of DNA methylation, which could explain many of the structural and functional changes seen in our model of epileptogenesis. The diversity of gene functions potentially affected by DNA methylation changes in our model are in line with the multifactorial nature of epilepsy and associated comorbidities and its link to brain development, function, and aging.To further investigate DNA methylation changes, a more permanent regulator for gene expression, we performed WGBS of cultures 7 days after initial precipitating injury. Principal component analysis and hierarchical clustering revealed a highly distinct pattern in genomic DNA methylation for glutamate- versus sham-treated neurons. Thereby, our data support extensive epigenomic changes even as short as 7 days after the treatment, confirming previous reports of DNA methylation dynamics in epileptogenesis ,43,56,57Characteristic genomic methylation signatures for different disease stages have been reported in various species, etiologies, and pathologies ,18,57,58


Enzymatic pretreatment of seeds is a novel approach that enhances the health benefits of the extracted oil. The study investigated the influence of the enzymatic pretreatment of seeds on the quality of oil from different pomegranate cultivars. The quality of the ultrasound-assisted (and ethanol-extracted) oil was studied, with respect to the refractive index (RI), yellowness index (YI), conjugated dienes (K232), peroxide value (PV) ρ-anisidine value (AV), total oxidation value (TOTOX), total carotenoid content (TCC), total phenolic compounds (TPC), fatty acid composition, phytosterol composition, ferric reducing antioxidant power (FRAP), and 2.2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging capacity. The seeds of three different pomegranate cultivars  were digested with an equal mixture of Pectinex Ultra SPL, Flavourzyme 100 L, and cellulase crude enzymes, at a concentration, pH, temperature, and time of 1.7%, 4.5, 40 °C, and 5 h, respectively. Enzymatic pretreatment of PS increased oil yield, PV, TPC, TCC, and DPPH radical scavenging capacity, but decreased the YI. The levels of K232, AV and TOTOX, fatty acids, phytosterols, RI, and FRAP, were not significantly affected by enzymatic pretreatment of PS. Principal component analysis (PCA) established that oil extracted from the ‘Acco’ seed after enzymatic pretreatment had higher yield, TPC, TCC, and DPPH radical scavenging capacity. Therefore, enzyme-pretreated ‘Acco’ pomegranate fruit seed is a source of quality seed oil with excellent antioxidant properties. Punica granatum L.) seed, a by-product of pomegranate fruit processed into juice and other products, is a rich source of pharmaceutical and nutraceutical compounds, such as tocopherols, sterols, carotenoids, polyphenols, and punicic acid . AsThe primary products of fatty acids oxidation are unstable and, therefore, can be further degraded to secondary products, such as the carbonyl compounds. Due to their low threshold values, and a major contribution to the development of rancid taste and flavour in seed oil, aldehydes are considered important compounds for the evaluation of secondary oxidation products in seed oil. The ρ-anisidine value (AV) measures the generation of aldehydes, primarily the 2-alkenals and 2.4-alkadienals from the decomposition of the hydroperoxides . The effTotal oxidation (TOTOX) value is used to estimate the overall oxidative degradation of fatty acids through the summation of PV and AV in fats and oils. It is clear in Apart from being natural food colour pigments, carotenoids possess biological properties that include antioxidant activity . In linePhenolic compounds have been linked with the reduction of certain chronic diseases, including cancer and cardiovascular diseases . Accordi3+-TPTZ) to the ferrous (Fe2+) state. PS enzymatic pretreatment significantly increased the DPPH radical scavenging capacity of ‘Acco’ oil extracts from 1.60 to 2.91 mmol Trolox/g PSO, but did not significantly increase the DPPH radical scavenging capacity of ‘Wonderful’ and ‘Herskawitz’ oil extracts, in spite of the significant increase in TPC and TCC , 2 h) of goldenberry seed did not significantly affect the phytosterols content of the extracted oil.On the other hand, the study of Fernandes et al.  reportedPS enzymatic pretreatment had a significant impact on PSO from ‘Wonderful’, whilst it showed an insignificant influence on ‘Herskawitz’ and ‘Acco’ oil extracts. For instance, punicic acid significantly decreased by 3.2% in ‘Wonderful’ oil extracts after PS enzymatic pretreatment. The finding that punicic acid significantly decreased after PS enzymatic pretreatment was unusual and an undesirable development since punicic acid is implicated with most of the PSO health benefits. Nevertheless, this could be linked to the significant increase in fatty acid oxidation after enzymatic pretreatment . On the 2/kg PSO) were lower than the limit in the Codex Alimentarius Commission (CODEX STAN 19-1981) standard for crude seed oil (15 meqO2/kg oil) [The dimension reduction property of principal component analysis (PCA) is valuable in exploring the treatments contributing more to the variation in the data. Therefore, to have a better understanding of cultivar and PS enzymatic pretreatment effect on oil quality, PCA was conducted on the multivariate data . The fir/kg oil) . F2, whiFresh good quality pomegranates  were harvested at commercial maturity  from Blydeverwacht farm, Wellington,  in Western Cape Province, South Africa, between February and April, during the 2019 season. Pomegranate seeds (PS) were separated from the juice and thoroughly washed with tap water prior to oven drying at 55 ± 2 °C for 24 h and kept at 4 ± 2 °C and 92 ± 3% RH until use ,57. Commv/v) concentration was prepared from the mixed enzymes in sodium citrate buffer at pH 4.5 and added to the samples. The enzyme suspension to seed ratio was kept constant at 900 µL/g (dry basis) in all experiments. The enzyme concentration and pH were established in a previous experiment using response surface methodology (RSM) (unpublished). The samples were hydrolysed at 40 ± 2 °C in a water bath for 5 h with continuous stirring at 150 rpm using an overhead stirrer , optimum conditions reported in prior research [w/w) moisture content [Uniformly ground pomegranate seed powder (<1 mm particle size) was thermally conditioned in an oven at 80 ± 2 °C for 10 min to deactivate the endogenous enzymes . Pectineresearch . The hyd content . n = 3) PSO samples were packed in brown bottles and stored at 4 ± 2 °C for further analyses [PSO from un-pretreated and enzyme pretreated seeds was extracted following the method described by Kaseke et al. . PS  with digital image recording . Specimens mounted on aluminium stubs were sputter-coated with a thin layer of gold (10 nm thick) using a gold sputter coater  to improve conductivity of the sample. The samples were examined at a voltage of 2 Kv.Refractive index at 25 °C was measured using a calibrated Abbe 5 refractometer . The yellowness index (YI) was calculated from lightness (L*) and yellowness (b*) values measured using a calibrated Chroma meter CR-410  .(1)YI=1PSO peroxide value (PV) was measured using the modified ferrous oxidation–xylenol orange (FOX) method described by Cruz et al. . Conjugaw/v) sodium carbonate and 6 mL of distilled water. After incubation in the dark for 30 min, the absorbances of the samples were measured at 760 nm using a UV Spectrophotometer . The results were reported as milligram gallic acid equivalent per g PSO (mg GAE/g PSO).The method of Ranjith et al.  was usedw/v) potassium hydroxide, 500 µL of 20% (w/v) ascorbic acid). An internal standard (100 µL of 5α-Cholestane (1000 mg/L) in chloroform) was added to each sample before the mixture was vortexed and incubated in an oven at 60 °C for 1 h. After cooling in ice for 10 min, distilled water (5 mL) and chloroform (2 mL) were added and the mixture vortexed and centrifuged at 3000 rpm for 4 min . The chloroform extracts (500 µL) were concentrated to ± 200 µL in 2 mL glass vials with a gentle stream of nitrogen. To 100 µL of the concentrated chloroform extracts, pyridine (100 µL), and N.O-Bis (trimethylsilyl) trifluoroacetamide (30 µL) were added. The mixture was vortexed before derivatization in an oven at 100 °C for 1 h. The GC–MS  was used to analyse the silylated sterol fractions. The samples were injected at 100 °C and temperature held for 2 min, before ramping to 250 °C at the speed of 7 °C/min and the temperature maintained for 2 min. Helium (1 mL/min) was the carrier gas and the split ratio of 5:1 and injection volume of 1.0 μL were used. The peaks were identified using a standard containing a mixture of sterols (β-sitosterol and stigmasterol). The retention times were used to identify the phytosterol compounds. The final results were reported as mg/100 g of PSO.The gas chromatography–mass spectrometry (GC–MS) method was applied to measure phytosterol composition . PSO (0.w/v) DPPH in 80% (v/v) methanol (freshly prepared). The vortexed samples were incubated in the dark for 60 min, after which the absorbance was measured at 517 nm using a UV spectrophotometer . A negative control of DPPH in 80% methanol was applied. The standard curve (5–100 mM) was prepared using Trolox and the results were expressed as mmol Trolox/g of PSO.The 2.2-diphenyl-1-picryl hydrazyl (DPPH) assay was used to measure the PSO antiradical activity . PSO . The results were reported as mmol Trolox/g of PSO.Ferric reducing antioxidant power (FRAP) of PSO was determined by mixing 40 µL methanol oil extracts, 200 µL distilled water, and 1.8 mL FRAP reagent . The FRAThe fatty acid methyl esters prepared according to Mphahlele et al. , were seData are presented as mean ± SD (standard deviation). Variables were compared by one-way analysis of variance (ANOVA) using Statistica software . Significant differences between means were determined using Duncan’s multiple range test. Microsoft Excel  was used for graphical presentations. The relationship between variables was determined by performing principal component analysis (PCA) using Microsoft Excel software .In conclusion, treating pomegranate seeds with enzymes before oil extraction is an alternative way to improve the oil yield and quality. The study showed that enzymatic pretreatment of PS enhanced oil yield, TPC, TCC and DPPH radical scavenging capacity, regardless of cultivar. This is desirable to the food and nutraceutical industries, with respect to improved profits and formulation of functional foods and nutraceutical products. Enzymatic pretreatment of seeds may not affect the quality of extracted oil with respect to K232, AV, and TOTOX. Moreover, fatty acids, phytosterols, and FRAP, were not significantly affected by enzymatic pretreatment of pomegranate seeds. Compared to ‘Wonderful’ and ‘Herskawitz’, ‘Acco’ pomegranate could be considered a preferable cultivar for seed and enzymatic pretreatment to improve yield and enhance antioxidant capacity of pomegranate seed oil. In this sense, the study demonstrated that PS enzymatic pretreatment improvement of oil quality is cultivar dependent.


Altogether, a new utilization mode for SBB is here proposed, as a means of efficient uranium sorption from UCW.Excess sludge (ES) treatment and that related to the uranium recovery from uranium-containing wastewater (UCW) are two hot topics in the field of environmental engineering. Sludge-based biochar (SBB) prepared from ES was used to recover uranium from UCW. Excellent effects were achieved when SBB was modified by acetic acid. Compared with SBB, acetic acid-modified SBB (ASBB) has shown three characteristics deserving interest: 1) high sorption efficiency, in which the sorption ratio of U(VI) was increased by as high as 35.0%; 2) fast sorption rate, as the equilibrium could be achieved within 5.0 min; 3) satisfied sorption/desorption behavior; as a matter of fact, the sorption rate of U(VI) could still be maintained at 93.0% during the test cycles. In addition, based on the test conditions and various characterization results, it emerged as a dual effect of acetic acid on the surface of SBB, i.e., to increase the porosity and add (−COOH) groups. It was revealed that U(VI) and −COO− combined in the surface aperture of ASBB  In addition, it also contains high concentrations of heavy metal ions, metal ions, and acid ions, such as sulfate ions and nitrate ions. This special industrial wastewater is mainly discharged by uranium mining or uranium hydrometallurgy. Generally, uranium presents in the valence form of U(IV) and U(VI). U(IV) does not dissolve in aqueous solution and usually forms precipitation, while U(VI) generally gives mobile aqueous complexes with COand OH−1 . Uraniumand OH−1 . In addiAt present, the main technologies for uranium recovered from UCW or UCW treatment could be summarized as chemical precipitation, ion exchange, membrane separation, biological treatment, solvent extraction, and sorption . Among t3O4-modified SBB also showed high uranium ion sorption efficiency (more than 90.0%)  was applied for biochar preparation . ES is mn 90.0%) . What isSo far, the most effective method for improving biochar sorption capacity or removal rate of heavy metals is to increase specific surface area or functional groups on its surface. For example, the effect of nitric acid on the surface area enlargement of biochar has been reported . In addiIn order to achieve the above requirements, the removal and recovery efficiency of uranium from UCW was comprehensively studied by involving acetic acid-modified SBB (ASBB) prepared from ES and acetic acid, including 1) differences in uranium recovery efficiency from UCW when SBB or ASBB were used, 2) impacts of variety factors  on uranium removal by ASBB, 3) kinetic and thermodynamic analysis of sorption, 4) ASBB uranium removal mechanism based on Brunauer–Emmett–Teller (BET), scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDS), Fourier-transform infrared spectroscopy (FTIR), and x-ray photoelectron spectroscopy (XPS) techniques. In this paper, acetic acid was used as a modifier to modify SBB and to treat UCW. This modification method could also be used to treat other heavy metal ions in the future.2ClCOOH), hydrochloric acid (HCl), ferrous sulfate heptahydrate (FeSO4.7H2O) and sodium hydroxide (NaOH), potassium hydroxide (KOH), and acetic acid (CH3COOH) were purchased from Sinopharm Group Pharmaceutical Co., Ltd. (Shanghai). Arsenio III [(HO)2C10H2(SO3H)2(N=NC6H4AsO3H2)2] and triuranium octoxide (U3O8) were purchased from Tianjin Kemio Chemical Reagent Co., Ltd., and China Academy of Metrology, respectively.ES was obtained from a WWTP located in Hengyang, China. The reagents used in this study were of analytical grade. Chloroacetic acid  was prepared by dissolving Uterature . All con3COOH solution (36.0%–38.0%) for 6 h and then washed with distilled water to neutrality, thus, eventually getting ASBB.The preparation process of SBB and ASBB is shown in Several values of reaction time , initial pH , adsorbent dosage , and initial uranium ion concentration  were scrutinized. U(VI) concentration in artificial UCW was kept at 10.0 mg/L except for particular cases. Dosage of SBB or ASBB was 0.30 g/L, and pH was 6.0.3+, Na+, Mg2+, Pb2+, and Cr6+. During the test, two dosages (0.3 and 0.5 g/L) of ASBB were set. HCl (0.01 mol/L) and NaOH (0.01 mol/L) were used for adjusting pH of artificial UCW.The desorption of ASBB was carried out by utilizing HCl (2.0 mol/L) as a desorption agent. The interference test of the sorption of U(VI) by coexisting ions (cation) in the solution was also carried out. Except for the solution containing 10.0 mg/L of U(VI), the concentration of coexisting ions in each solution was simulated to 10.0 mg/L. The interfering ions involved were FeU(VI) concentration was determined by Arsenazo III spectrophotometer . The absqe (mg/g) and removal rate η for each sorbent (SBB or ASBB) were calculated according to c0 and ce are the initial and equilibrium concentrations of uranium in the solution, v is the solution volume, and m is the mass of adsorbent. Langmuir and Freundlich sorption isotherm models were introduced to fit the U(VI) sorption data for ASBB under equilibrium conditions , while KF and n are the Freundlich parameters, respectively, representing the sorption capacity and the sorption intensity.The uranium equilibrium specific sorption capacity nditions , 4. The nditions :qe=qmKLqt refers to the specific sorption capacity at t time, κ1 is the PFO sorption rate constant (min−1), and κ2 is the PSO sorption rate constant (g/mg·min−1).In order to investigate in detail the U(VI) sorption process by ASBB, kinetic data were fitted by pseudo-first-order , the contents of –OH, –COO, and –COOH were determined by the Boehm method . Three sCO2 = 10–3.5 atm under pH from 3.0 to 9.0 were simulated by Visual MINTEQ 3.1 (via Casa XPS software (Version 2.3.13).The existing forms of uranium in UCW (10.0 mg/L) and PNTEQ 3.1 . The speF values of MSK, CTE, CTI, and AT were 87.09, 9.84, 4.34, and 1.18, respectively,  removal is presented in refer to . The p-vtendency . In addi22+, (UO2)2(OH)22+, (UO2)2OH3+, (UO2)3(OH)5+, (UO2)3(OH)42+, (UO2)4(OH)7+, UO2(CO3)34−, and (UO2)2CO3(OH)3−. The U(VI) removal rate was only 42.4% when pH = 3.0, where uranium mainly exists in the form of UO22+ in UCW. Because the solution pH value was in this case too low, a lot of H+ competed with UO22+ sorption. Meanwhile, an H+ proton layer on the surface of ASBB could be formed, rather than UO22+. The electrostatic repulsion of ASBB to UO22+ might increase; thus, the removal rate of uranium was relatively low (2)2(OH)22+, (UO2)3(OH)5+, (UO2)4(OH)7+. The low protonation degree of these forms favored the sorption of uranium by ASBB (.) might be gradually assembled on the surface of ASBB. H+ on these groups then decreased, so the electronegativity of these groups increased. The binding ability and reaction probability between functional groups and uranium increased due to this phenomenon. The uranium removal rate increased under this condition. When pH was 6.0, the U(VI) removal rate peak was 97.2%. When pH was between 7.0 and 9.0, the uranium was mainly in the form of UO2(CO3)34− and (UO2)2CO3(OH)3−. These forms were difficult to be adsorbed by ASBB, and the removal rate of uranium was reduced. Therefore, pH = 6.0 was suggested as the optimal condition for U(VI) sorption from UCW by ASBB.vely low . When th by ASBB . With th+ through ion exchange  could be replaced by Hexchange , and theexchange . Figure + had little effect on the removal of U(VI) by ASBB, while it would be inhibited by Mg2+, Pb2+, and Cr6+. In particular, Cr6+ had the greatest impact on ASBB’s sorption of U(VI). The main reason for this phenomenon might be the competitive sorption of these ions and U(VI) on the surface of ASBB. Unlike these ions, Fe3+ facilitated the U(VI) removal. The main reason might be that when pH = 6, Fe3+ could be hydrolyzed into Fe(OH)3 colloids .colloids , and U(Vloids (3 . As a reR2 was 0.998 (PFO) and 0.997 (PSO), indicating that both physical and chemical sorption occurred during the sorption of U(VI) by ASBB.qm = 178.194 mg/g (ASBB adsorbent), a value consistent with the laboratory result qm = 179.88 mg/g. The correlation coefficient was 0.943 (Langmuir model) and 0.989 (Freundlich model), indicating that the sorption of U(VI) by ASBB was mostly dominated by multilayer sorption.The results of the thermodynamic analysis are illustrated in The microstructure and surface elements of SBB, ASBB, and ASBB-U  were characterized by SEM and EDS . As showAccording to 4 hysteresis curve  of ASBB increased with respect to the untreated biochar. Namely, a pore expansion function of acetic acid was observed. Then SSA for ASBB-U decreased. It indicates that uranyl ion was adsorbed in the pores of the ASBB surface. When the pores were blocked by the absorbed uranyl ion, the SSA of the adsorbent obviously decreased.−1), –COO , –Si–O–Si (781 cm−1), and -CCl4 (476 cm−1) were the main groups retrieved on the surface of SBB, ASBB, and ASBB-U. When the FTIR spectra of SBB, ASBB, and ASBB-U are compared, it is seen that –COO  was found on the ASBB surface as a new group with respect to SBB, indicating the modification of SBB by acetic acid. Moreover, when uranium was adsorbed on ASBB, some of the peak’s position and intensity changed. The peak of –OH stretching vibration at 1,331.37 cm−1 disappeared, indicating that –OH might react with U(VI) by deprotonation. In addition, the symmetric stretching vibration peak of –COO at 1,406.32 cm−1 moved to 1,384.53 cm−1. Although the peak shape was stable, its intensity was enhanced. The difference of the stretching vibration frequency between –COO antisymmetric stretching vibration peak  and –COO symmetric stretching vibration peak  was more than 200 cm−1 (233.29 cm−1). This indicates that –COO and U(VI) were combined in monodentate coordination mode  on ASBB. Results are illustrated in ion mode .2/5  corresponded to U(VI) on the surface of ASBB-U 3(OH)5+ and (UO2)4(OH)7+. They were combined with –COO in monodentate coordination. The specific equation reads , and relevant references , the mecon reads :(7)Excess sludge (ES) and acetic acid were utilized to obtain a robust adsorbent starting from sludge-based biochar (SBB), for U(VI) abatement in uranium-containing wastewater (UCW). Compared with SBB, the removal efficiency and sorption capacity of the acetic acid-modified biochar (ASBB) could be effectively improved. An optimal U(VI) removal rate of 97.8% could be achieved, while initial conditions were pH = 6.0, U(VI) = 10.0 mg/L , adsorbent dosage = 0.30 g/L, and sorption time = 5.0 min. The beneficial effect was attributed to the double action of expanding pores and increasing –COOH functional groups following the acetic acid modification treatment. The process of U(VI) sorption by modified biochar relies on both physical and chemical sorption. The U(VI) removal mechanism by ASBB was of monodentate coordination binding between –COO– and uranium. In addition, ASBB had good reusable performance. Hence, the quick sorption and outstanding efficiency of ASBB offer a meaningful support for the use of biochar in uranium recovery from UCW and for reutilization of ES.