Brain region specific regulation of anandamide (down) and sphingosine-1- phosphate (up) in association with anxiety (AEA) and resilience (S1P) in a mouse model of chronic unpredictable mild stress

Brain region specific regulation of anandamide (down) and sphingosine-1- phosphate (up) in association with anxiety (AEA) and resilience (S1P) in a mouse model of chronic unpredictable mild stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Brain region specific regulation of anandamide (down) and sphingosine-1- phosphate (up) in association with anxiety (AEA) and resilience (S1P) in a mouse model of chronic unpredictable mild stress Caroline Fischer, Dominique Thomas, Robert Gurke, Irmgard Tegeder This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4408665/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Aug, 2024 Read the published version in Pflügers Archiv - European Journal of Physiology → Version 1 posted 5 You are reading this latest preprint version Abstract Chronic unpredictable and unavoidable stress is associated with mental health problems such as depression and anxiety, whereas cycles of stress and stress relief strengthen resilience. It has been suggested that increased breakdown of brain endocannabinoids (eCB) promotes a feeling of adversity. To assess the impact of stress on bioactive lipid homeostasis we analyzed eCB, sphingolipids and ceramides in seven brain regions and plasma in a mouse model of chronic unpredictable mild stress. CUMS was associated with low levels of anandamide in hippocampus and prefrontal cortex in association with indicators of anxiety (elevated plus maze). Oppositely, CUMS caused elevated levels of sphingosine-1-phosphate (S1P d18:1) and sphinganine-1-phosphate (S1P d18:0) in midbrain and thalamus, which was associated with readouts of increased stress resilience, i.e., marble burying and struggling in the tail suspension tests. In the periphery, elevated plasma levels of ceramides revealed similarities with human major depression and suggested unfavorable effects of stress on metabolism, but plasma lipids were not associated with body weight, sucrose consumption or behavioral features of depression or anxiety. The observed brain site specific lipid changes suggest that the forebrain succumbs to adverse stress effects while the midbrain takes up defensive adjustments. Sphingolipids endocannabinoids chronic unpredictable mild stress ceramides hippocampus prefrontal cortex thalamus midbrain anxiety depression resilience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Stress exposure is normal in daily life, but it is also a predisposing risk factor for metabolic, cardiovascular, and mental disease such as anxiety and depression [ 1 – 3 ]. Stressors do not influence all individuals equally. Some are vulnerable while others are more resilient [ 4 , 5 ]. The adaptations of the brain to acute and short-lasting stressful conditions involves neuronal systems that also process rewarding stimuli [ 6 – 8 ]. Studies indicate that the ability to cope with acute or chronic stress relies in part on the neurotransmitter, dopamine, mostly considered as a signal of reward or stress relief [ 4 , 9 , 10 ]. However, dopamine is also involved in aversive motivation [ 11 , 12 ]. Acute stress increases extracellular dopamine levels in the mesolimbic mesocortical dopamine system, particularly nucleus accumbens and prefrontal cortex (PFC) [ 6 , 13 ], but chronic or intense stressors, particularly prolonged, repetitive, unpredictable and unavoidable stress rather inhibits dopamine release or dopamine receptor responsiveness [ 9 , 14 – 16 ]. In rodents, chronic restraint stress, chronic social defeat stress, and chronic unpredictable mild stress (CUMS) are associated with depressive-like behaviors [ 17 , 18 ] and are linked to suppressed midbrain dopaminergic neuronal activity [ 6 , 11 ]. There is some evidence from previous studies that microbiome derived lipids [ 19 ] and endogenous bioactive lipids including prostaglandins, sphingolipids and endocannabinoids contribute to the mal-adaptations to chronic stress at the molecular level [ 20 – 22 ], and in turn, are regulated by chronic or acute stress [ 23 , 24 ]. Ceramides are believed to aggravate or maintain aversiveness [ 21 ] and have been suggested as putative targets for antidepressants [ 25 – 27 ] whereas endocannabinoids (eCBs) protect against psychological sequelae of chronic stress [ 28 ], likely as positive indirect modulators of the dopamine system. DA neurons in the midbrain receive excitatory glutamatergic and inhibitory GABAergic input [ 29 – 31 ], and both are under inhibitory control via presynaptic cannabinoid receptors [ 32 – 35 ]. Functions of sphingoid-base sphingolipids are unclear and mostly based on studies with FTY720 (fingolimod [ 36 ]), which acts as a sphingosine-1-phosphate (S1P) receptor agonist after phosphorylation but leads to receptor downregulation. One study revealed that S1P-dependent stress resilience required signaling through S1P receptor 3 ( S1PR3 ) in medial prefrontal cortex [ 37 ], but alternative effects such as inhibition of microglial inflammasome activation were also suggested [ 38 ]. The evidence of a mutual endocannabinoid-mediated stress-lipid signaling arises form preclinical studies with genetic ablation or pharmacologic antagonism of the cannabinoid type 1 receptor ( Cnr1 , CB1) [ 39 – 41 ], or from clinical experience with the CB1-antagonist, rimonabant [ 42 ]. Knockout of CB1 results in exaggerated neuroendocrine and behavioral responses to acute stress including anxiety, reward sensitivity, pain, morphologic changes in the amygdala, and hippocampal synaptic plasticity [ 40 , 43 – 47 ]. Under acute stress, endocannabinoids limit the magnitude of the stress response and facilitate recovery after cessation of stress exposure [ 45 , 48 ]. However, under chronic or severe stress, the endocannabinoid system appears to “collapse” in the sense that CB1 receptors are downregulated or dysfunctional or that endocannabinoids are missing [ 49 ]. Hence, the ability to modulate the synaptic release of neurotransmitters such as glutamate and gamma-aminobutyric acid is lost [ 50 – 52 ]. Consequently, inhibitors of endocannabinoid breakdown attenuated stress evoked behavioral manifestations of depression or anxiety [ 48 , 53 – 55 ]. In humans, posttraumatic stress disorder or major depressive disorder is associated with reductions of the circulating levels of endocannabinoids [ 56 – 58 ], and a significant proportion of individuals using the CB1 antagonist rimonabant for weight loss developed indices of anxiety and depression and suicide and finally, withdrawal of the drug [ 59 , 60 ]. Based on these studies we hypothesized that chronic stress leads to brain-region dependent changes of endocannabinoids, which in turn would increase the suffering from chronic stress. The vicious cycle may be fixated by genetic or epigenetic modifications and further profound changes of connected or independent lipid signaling paths that as a whole determine the behavioral outcome and susceptibility to mental disease on one side and gain of resilience or even reward upon stress relief on the other side. To address this hypothesis, we analyzed lipids of emotion-relevant classes (endocannabinoids, ceramides, hexosylceramides and sphingoid bases) in seven brain regions and plasma in association with behavior in a model of chronic unpredictable mild stress in mice. Methods Mice Animal studies are reported and were conducted in compliance with the ARRIVE guidelines [ 61 ]. The experiments were approved by the local Ethics Committee for Animal Research (Darmstadt, Germany V 54 − 19 c20/15 FK1074) and adhered to the European guidelines and to those of GV-SOLAS for animal welfare in science. We used female mice which were raised in the local breeding facility. Matched pairs according to body weight and age were submitted to chronic unpredictable mild stress (CUMS; n = 11) and control groups (n = 10). Group assignment was blinded. The ages at the start ranged from 6–15 weeks (average 9.5 weeks) in both groups. The average weight at onset was 20.5 ± 0.7 g (CUMS) and 20.6 ± 1.2 g (control). Mice were housed in pairs (except one cage with n = 3) and kept in a controlled environment (12-hour dark/light cycle, 23°C, 55% humidity) with food and water ad libitum. Animal numbers were estimated by power analysis using GPower [ 62 ]. The probability of type-1 error was set to α = 0.05, and type-2 error was set at β = 0.2. Chronic unpredictable mild stress (CUMS) To evoked mild stress mice are exposed daily to different types of mild stressors, such as temporary isolation or crowded housing, tilted home cages, wet bedding, predator odor, cold exposure, restraint, itch, no food, no water or disrupted dark-light cycle [ 63 , 64 ]. The time of each stressor varies from 0.5-4 h per day or overnight, and the protocol can last for 3–12 weeks. The detailed protocol used in this study is provided as Supplementary timetable. To integrate behavioral tests of control mice the stress protocol lasted for 6 weeks with a short break of 6 days without stressors. Behavioral tests of CUMS mice were integrated in the last stress exposure days. The stressors included cage switch, itch, cold exposure, TMT odor, headache (nitroglycerin i.p.), no bedding, wet bedding, lights on overnight, no food overnight, no water overnight, restraint, cage shaking, tilted cage, ultrasound noise. The behavior tests using tail suspension and elevated plus maze per se are also considered as stressors. Elevated Plus Maze (EPM) The EPM takes advantage of the physiologic self-protective hiding of mice in the dark versus their curiosity. The test is considered to measure anxiety-like behavior. The standard EPM maze was configured with two orthogonally arranged closed arms (25L × 5W × 15H cm) and two open arms (25L × 5W × 0.3H cm) connected by a central open square (5 × 5 cm), and it was elevated 60 cm above the floor and illuminated from above. The floor and walls were made of grey PVC. The maze was placed in a quiet enclosure of the test room and mice were habituated before start. At test start, mice were placed individually into the center platform facing an open arm and were allowed to move freely for 10 min [ 65 ]. The behavior was video recorded with a camera mounted above the maze. VideoMot2 (TSE Systems GmbH, Bad Homburg, Germany) was used for automatic tracking and analysis of arm entries, times in closed/open and distances. The anxiety index was calculated for as AI = (time in closed/total time in arms) – (time in open/total time in arms). Tail Suspension Test (TST) In the TST mice are suspended by the tail, which elicits defensive struggling and immobile hanging. The latter is often interpreted as resignation or depression resulting from an unsolvable and aversive situation [ 66 ], and the time of immobility the primary readout. We used a self-constructed observation chamber which was divided into five compartments, separated by dark grey spacers, each compartment with hook 30 cm above the floor. A widefield camera was mounted in front on a tripod to capture five mice simultaneously. The tail was fixed with adhesive tape to the hook, and each test lasted 10 min [ 67 ]. The video recordings were analyzed posthoc by using a computer key to measure the time and was done without knowledge of the group assignment. Sucrose preference and latency Mice are highly addicted to sweet water. Compared with daily tap water volumes of about 4 ml, they may consume up to 16 ml sweet water (2% sucrose). High sucrose preference may be interpreted as consolation, or compulsiveness/addiction, depending on the extent and choices. To measure sucrose preference, a 2-choice test was used. Mice were housed in pairs per cage and habituated for 3 days by providing bottles of tap water on the right and left side of the cage. One bottle was then replaced with 5% sucrose in tap water, randomly on the right or left side. The volume intake was assessed by daily weighing the bottles. The latency to first licking of sucrose-water after an overnight water restriction was assessed in the home cage and in a new cage with new bedding. Mice were tested individually. The latency was assessed by observation with a stopwatch. Marble burying The marble burying test has been used to assess stress or novelty evoked anxiety (defensive burying or neophobia burying) [ 68 , 69 ] or assessment of repetitive and compulsive behavior [ 70 ]. The test takes advantage of the normal spontaneous burying and digging behavior. Increased marble burying under stress reflects an inherent defensive response aimed at protecting from harmful objects. The behavioral meaning of marble burying is however debatable [ 68 , 71 ]. For the MBT, 12 neutral 0.5-inch glass marbles were arrayed on the surface of clean, thick 7 cm bedding. The number of buried marbles (covered at least ¾ with bedding) was counted at 5 min, 10 min, 20 min and at the end of a 30 min observation period. The MBT was done during and after completion of the CUMS protocol. The 30 min end point of "MBT-during-CUMS" was used for lipid association analyses. The investigator was unaware of the group assignment. Tissue and blood sampling Mice were euthanized by CO2 in a chamber with adjustable stepwise increasing flow. Blood samples were collected in 500 µl K3-EDTA microtubes (Microvette Sarstedt) after cessation of respiration via cardiac puncture with a 27G needle attached to a 1 ml syringe. The samples were immediately centrifugated in a mini- tabletop centrifuge at 2000 g for 3 min, plasma transferred into microtubes using a 100 µl pipette and directly frozen in liquid nitrogen and kept at -80°C until analysis. The brain war rapidly removed, olfactory bulb removed (discarded), cerebellum collected, weighed and frozen in liquid nitrogen, then brain cut sagittal, the halves unfolded, and then regions collected from rostral to dorsal: orbitofrontal and dorsal prefrontal cortex, striatum including nucleus accumbens, hippocampus, thalamus, hypothalamus, and midbrain. All samples were weighed on a precision scale and directly frozen in liquid nitrogen and kept at -80°C until analysis. Lipid concentrations are normalized on mg of tissue. Orbitofrontal and dorsal prefrontal cortex samples were analyzed separately, but concentrations were then averaged to get one PFC-value per mouse to reduce brain sites and gain power. Analysis of lipid signaling molecules Bioactive lipids including sphingoid bases and ceramides, lysophosphatidic acids (plasma only), and endocannabinoids (eCBs) were analyzed in plasma and in brain tissue homogenates using liquid-liquid-extraction (LLE) followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) as described in detail in a previous study [ 26 ]. Briefly, brain tissue samples were homogenized in ethanol:water (1:3, v/v) using a Mixer Mill MM400 (Retsch, Haan, Germany). Afterwards, brain tissue homogenates as well as plasma samples were extracted using an LLE protocol as described in detail in the previous publication. Sphingolipids were separated using an Agilent 1200 HPLC system equipped with a Zorbax C18 Eclipse Plus UHPLC column (50 × 2.1 mm, 1.8 µm, Agilent technologies, Waldbronn, Germany). The analysis of LPAs (plasma only) was done on the same HPLC system using a Luna C18 column (50 × 2.0 mm, 5 µm, Phenomenex, Aschaffenburg, Germany). For the chromatographic separation of endocannabinoids an Agilent 1290 Infinity I UHPLC system equipped with an Acquity UPLC BEH C18 UPLC column (100 × 2.1 mm, 1.7 µm, Waters, Eschborn, Germany) was used. The quantification of all analytes was performed using a hybrid triple quadrupole-ion trap mass spectrometer QTRAP 5500 or 6500+ (Sciex, Darmstadt, Germany) equipped with a Turbo-V-source operating in positive ESI mode for sphingolipids and endocannabinoids and in negative ESI mode for LPAs. Quality control samples of three different concentration levels (low, middle, high) were run as initial and final samples of each run. For all analytes, the concentrations of the calibration standards, quality controls and samples were evaluated by Analyst software 1.6 and MultiQuant software 3.0 (Sciex, Darmstadt Germany) using the internal standard method (isotope-dilution mass spectrometry). Calibration curves were calculated by linear or quadratic regression with 1/x weighting or 1/x2 weighting. To assess putative biases caused by sample sequence, concentrations were plotted versus analysis number. For ethanolamide endocannabinoids (AEA, OEA, PEA) linear regression analysis revealed a shallow but significant linear raise (significantly different from zero) with the sample number. The concentrations were therefore adjusted according to analysis sequence number and the slope of the regression line. There was no effect of sample sequence for any of the other analytes. Statistics Lipid concentrations are presented as scatter plots with mean ± standard deviation (SD) or box-scatter plots, where the box is the interquartile range and the whiskers show minimum to maximum, or the 95% confidence interval (CI), specified in the figure legend. Data were analyzed with SPSS 29, Origin Pro 2024 and GraphPad Prism 9.0. Principal component analysis (PCA) and partial least square analysis (PLS) were used to reduce dimensionality and identify the factors which contributed most to the difference between treatment groups and brain sites. In addition, linear canonical discriminant analysis (DA) was used to assess the predictability of group membership based on DA scores. DA was performed without and with bootstrapping, the latter using a stratified random sampling approach considering gender and age, and 100 iterations. Lipid concentrations and behavioral readouts were compared between groups using analyses of variance (two-way ANOVA for brain site X treatment (CUMS or control), or t-tests according to the data subgroup structure and distribution. In case of significant results of ANOVAs, treatment groups were compared per brain site using t-tests comparing CUMS versus control. P-values were adjusted according to Šidák for multiple comparisons or were not adjusted if only two groups were compared. For behavioral time course data, 2-way ANOVA for repeated measurements "time" X "group" was used. The alpha level was set at 0.05 for all comparisons and asterisks in the figures refer to adjusted P-values. For cluster analyses and polar plots, lipid concentrations were normalized to the median of all samples of the respective lipid to allow for a combined analysis and presentation. For correlation plots, lipids and behavioral readouts were scaled and correlations are coded by color (blue negative correlation, red positive correlation) and bubble size according to the adjusted R-square. Further analyses consisted in multiple regression analyses to reveal associations between behavioral readouts and lipids that were different between CUMS and control mice at one or more brain sites. The regression analysis only included the CUMS groups based on the hypothesis that an increase or decrease of the respective lipid would be associated with a change of behavior rather than that the lipid concentration as such would predict a behavior. There was no association of behavior for any lipid in the control group. Results CUMS evoked mild weight loss and anxiety As expected CUMS was associated with mild effects on health and behavior (Fig. 1 A-H) manifesting in a mild reduction of the body weight (Fig. 1 A), increase of plasma ceramides (Fig. 2 ) and decrease of the time spent in open arms of the EPM (1D, E), which may be interpreted as anxiety-like behavior. In addition, the latency to first licks of sweet water was increased in an unfamiliar novel cage as compared to controls (Fig. 1 G), but sucrose intake was not different (Fig. 1 H). The behavior agrees with anxiety-like behavior ("neophobia") rather than "depression". There was no difference in marble burying behavior (Fig. 1 B) and total distance moved in maze tests (Fig. 1 F). In the TST, immobility time was reduced in the first trial showing that CUMS mice spent more time with struggling but was not consistently maintained upon repeated testing (Fig. 1 C). Plasma concentrations of endocannabinoids and sphingolipids were analyzed to assess the systemic metabolic impact of CUMS (Fig. 2 ). Ceramides were increased, particularly Cer d18:1/16:0 and Cer d18:1/22:0 which agrees with the expectation that stress disrupts metabolic homeostasis. High plasma concentrations of ceramides have been associated with obesity, diabetes [ 72 ] and mental disease (major depression, bipolar disorder) [ 26 ]. In addition, endocannabinoids, OEA and PEA were increased, likely elicited by the loss of body weight during CUMS and favoring rapid body weight regain after CUMS (sample were taking after BW recovery) [ 73 ]. Brain regions have site-specific lipid patterns Lipid species of four classes (4x endocannabinoids, 4x sphingoid bases, 4x ceramides, 5x hexosylceramides) of seven brain sites were submitted to linear canonical discrimination analysis to reveal brain site specific patterns and effects of CUMS (Fig. 3 ). Scatter plots of the discriminant scores (canonical variable 1 versus 2) show that and lipid patterns of midbrain (MB) and thalamus (Th) are similar, that the cortical patterns of PFC and hippocampus are closely related, and striatum is highly variable in between. The cerebellum is unique in its lipid patterns. The discrimination task was to separate sites and CUMS levels were used for learning and applied to controls. The analysis shows that the patterns apply to both groups in agreement with the expectation of the model, which was meant to cause mild stress but no profound disruption. The effects CUMS however do reveal at the level of individual lipid species and are site-specific. In the first set of analyses presented as circular plots (Fig. 4 ) data were transformed to percentages versus overall median to visualize the effect of CUMS. The forebrain to midbrain (top to bottom) the CUMS group impresses with reduced anandamide and lactosylceramides in PFC, increase of sphingoid bases and ceramides in striatum and a further increase of sphingoid bases (but not ceramides) in thalamus and midbrain. Across brain regions, S1P d18:1 (sphingosine-1-phosphate) and S1P d18:0 (sphinganine-1-phosphate) are higher in the CUMS group. Correlation of lipids across brain regions with behavioral readouts Lipid levels across brain regions (i.e., the average of all sites for each mouse), body weights and behavioral readouts were submitted to correlation analyses and plotted as correlation plots (Fig. 5 ) to reveal if and how brain lipids or behavioral features were associated with each other. The analysis was done for control and CUMS separately to compare patterns. The CUMS correlation map has more positive (red) and negative (blue) correlations. Again, S1P and LacCer show the strongest differences. In controls, S1P is negatively correlated with ceramides, which is lost or inverted in CUMS. In CUMS there are strong negative correlations of AEA with behavior which is not evident in controls. Correlation maps, circular lipid plots and behavior suggested two hypotheses (i) anandamide (AEA) deficiency in hippocampus and cortex evoked by CUMS is associated with anxiety and (ii) S1P in subcortical structures is associated with defensive and combative depression-averting behavior. To address the hypothesis individual site-specific lipid concentrations were analyzed in detail in associations with behavioral features using multiple regression analyses (Figs. 6 – 9 ). CUMS-evoked low AEA in cortex associated with anxiety AEA was significantly reduced in the PFC in CUMS mice versus controls, and OEA was increased in the hippocampus (Fig. 6 A). As revealed in Fig. 2 , OEA and PEA were increased in plasma of CUMS versus control mice. AEA is a canonical cannabinoid receptor ligand, and therefore associations with behavior were primarily assessed for AEA. The hippocampus was chosen as primary site (although n.s.) because the HC concentrations in CUMS mice showed a broad range suggesting that HC-AEA may reflect the individual susceptibility to CUMS evoked behavior. Linear regression analyses revealed that low AEA in hippocampus (or PFC not shown) was significantly associated with long distance (Fig. 6 B) (or long time, not shown) in the closed arms in EPM. The lower AEA, the more time was spent in the closed arm, suggesting that low AEA increased anxiety or reduced curiosity. Low AEA in the hippocampus was also associated with a long sucrose latency in an unfamiliar cage, which however did not reach statistical significance (P 0.062). CUMS-evoked high S1P in thalamus and midbrain associated with TST struggling and MBT S1P d18:1 (sphingosine-1-phosphate) and S1P d18:0 (sphinganine-1-phosphate) were increased in midbrain and thalamus in CUMS mice (Fig. 7 ). In addition, non-phosphorylated sphinganine was increased in hippocampus, PFC, and cerebellum in CUMS versus control. Linear regression analyses (Fig. 8 ) show that high S1P d18:0 (sphinganine-1-phosphate) is associated with long paths in the closed EPM arms (Fig. 8 A). However, there was no association with time spent in closed arms (not shown, linear adj R-square − 0.111; ANOVA P-value 0.958) showing that mice with high S1P d18:0 in midbrain were more active within the closed arms. There was no association of S1P d18:0 with sucrose latency (Fig. 8 B). S1P d18:1 in the thalamus (or across regions, not shown) was associated with a high number of buried marbles in the MBT (Fig. 8 C) and high struggling time in TST (which is the inverse of the immobility time) (Fig. 8 D). Although previous studies in mice suggest that ceramides are particularly important for stress-evoked depression like behavior and plasma ceramides agree with the expected stress evoked increase, there were no consistent between group differences, and no significant association between plasma and across-brain ceramides levels. Notably, ceramides were high in striatum in both groups compared with other brain regions. Discussion The present study shows that CUMS in mice leads to mostly subtle site-specific adjustments of endocannabinoids, particularly a loss of AEA in the PFC and increase of sphingolipids, particularly S1P in midbrain and thalamus. Low AEA in PFC and hippocampus are associated with anxiety-like behavior i.e., preference of dark zone in EPM and long sucrose-latency in an unfamiliar cage. These results agree well with previous studies of chronic stress in mice where AEA was found reduced in the brain, albeit not specifically in cortical structures but throughout studied brain regions [ 28 , 55 , 74 , 75 ]. Other studies using electrophysiology and CB1 receptor knockout have suggested that specifically the endocannabinoid systems of the hippocampus and amygdala have a key role in anxiety extinction [ 52 , 76 , 77 ] and stress relief [ 78 ]. Human imaging studies using cannabinoid radioligands reveal increases of free CB1 binding capacity in stress associated diseases such as posttraumatic stress disorder suggesting a relative deficiency of endocannabinoids at affected sites [ 79 , 80 ]. Brain samples in our study were collected directly at the end of the CUMS protocol, hence not allowing mice to learn that the stress period was finished. Therefore, we assume that the brain was captured in a state of heightened alertness, unease and fear for the next stressor and not in a state of stress relief, which is expected to switch the brain into reward-mode [ 4 , 5 ] associated with an increase of endocannabinoids and enhancement of dopamine release from crucial reward sites of the mesolimbic system [ 81 – 84 ]. The repeated experience of stress relief was shown to increase stress resilience and prevent depressive symptoms in mice [ 4 ]. Resilience in mice is mostly inferred from non-occurrence of stress-evoked fear i.e., as a negative readout, and the meaning of specific behaviors is context-sensitive and complex. In the present study we interpreted the struggling behavior in the TST and the marble burying behavior in the MBT as positive readouts of active defensive behavior and hence resilience [ 71 , 85 ], as opposing to depression that would manifest as immobility, low locomotion, reduced feeding or reduced nest building [ 66 , 86 ]. This interpretation is supported by studies which use the immobility time in TST or small numbers of buried marbles as indicators of depression [ 17 , 87 – 89 ]. The MBT is particularly controversial. Digging and burying of noxious, harmless, or rewarding objects is part of normal food searching and nest building behavior, and it is expressed in home cages without stress and under anxiogenic circumstances for example imposed by exposure to noxious objects or predator odor [ 71 ]. Defensive burying has been defined as the process of moving bedding material to cover harmful stimuli such as sources of electrical foot shock, and it is used as a measure of aversive anxiety [ 69 , 90 , 91 ]. In addition, burying of novel objects under stressful conditions is considered as neophobia but mostly does not well agree with other readouts of novelty-evoked fear [ 71 ], and excessive marble burying was suggested to reveal nonfunctional repetitive behavior analogous to behavioral symptoms of obsessive–compulsive disorder [ 68 , 92 , 93 ], impulsivity, autism, and dementia, however with low predictive value for therapeutics of such disorders [ 94 ]. Hence, as a stand-alone test, MBT offers alternative interpretations. In the present study, the numbers of buried marbles in CUMS mice were more variable but not significantly different from those of control mice but were positively correlated with the struggling time in the TST, which is the inverse of the immobility time, and with S1P d18:1 and S1P d18:0 levels in the thalamus and across brain regions. It is not much known about putative functions of S1P in the brain for adjustments of the brain towards chronic stress and coping with chronic stress. However, one study revealed that fingolimod increased stress resilience (i.e., prevented stress-evoked depression-like behavior) in a model of chronic unpredictable stress via activation of S1PR3 in the medial prefrontal cortex [ 36 , 37 ]. Once phosphorylated, fingolimod is an S1P receptor agonist [ 95 ]. However, it is mostly recognized for opposite net effects, i.e., reduction of S1P signaling resulting from receptor downregulation. The latter effect prevents T-cells from egress of secondary lymphoid organs, hence explaining its therapeutic efficacy in autoimmune-mediated diseases, particularly preventing relapse in multiple sclerosis [ 96 ]. Major depression, schizophrenia, PTSD, and other psychiatric diseases are believed to be contributed or sustained by inadequate immune activation [ 97 – 100 ]. Hence, many studies assessed the efficacy of fingolimod in experimental models of such psychiatric diseases mostly with some therapeutic benefit for fingolimod treated mice [ 95 , 101 – 104 ], which was however not confirmed in clinical studies [ 105 ]. Considering differences in treatment schedules in mice and psychiatric patients, it may be suggested that long-term fingolimod leads to receptor downregulation in the brain similar to its regulation in immune cells and hence, interferes with resilience strengthening effects of endogenous brain S1P under stress conditions. Indeed, depression is frequent in MS patients receiving fingolimod, but switching to fingolimod from other disease modifying drugs was reported to reduce depression at least temporarily [ 106 ]. Although chronic stress evoked mental health issues are believed to be contributed by immune activation, we did not observe an increase of plasma S1P in CUMS mice. Instead, plasma lipids revealed an increase of ceramides, particularly Cer d18:1/22:0 which are increased in patients with major depression and bipolar disorder, again particularly Cer d18:1/22:0 [ 26 , 107 ]. Ceramides were not associated with body weight presumably because time points of blood sampling and weight loss during CUMS did not match. Blood was obtained at the end of the CUMS protocol when the body weight was already restored. Nevertheless, alterations of plasma ceramides well agree with the concept of stress-evoked unfavorable metabolic effects which however did not correlate with behavioral readouts of "mental" health in our mice. In summary, we show that CUMS in mice resulted in low AEA levels in cortical brain regions in correlation with anxiety-like behavior, and high S1P levels in midbrain and thalamus in correlation with defensive behavior. The results agree with previous studies of brain anandamide under stress and strengthen the idea that cannabinoids might be useful in certain cases of PTSD [ 108 , 109 ]. S1P results are novel. They agree with strengthening of resilience with short-term fingolimod treatment in mice but suggest that sphingosine kinase inhibitors that are under investigation for cancer and fibrosis [ 110 – 113 ] might affect mental health, so far not observed. Abbreviations AG arachidonoylglycerol AEA anandamide Cer Ceramides LacCer Lactosylceramides GlcCer glucosylceramides eCB endocannabinoid SPH sphingolipids S1P d18:1 sphingosine-1-phosphate S1P d18:0 Sphinganine-1-Phosphate CUMS chronic unpredictable mild stress EPM elevated plus maze SPT sucrose preference tests TST tail suspension test ScrLat Sucrose licking latency Declarations Article dedication The article is dedicated to our colleague, Andrea Huwiler, who recently passed away before the Special issue could be finalized. Funding The study was supported by the Deutsche Forschungsgemeinschaft (DFG) via the collaborative research center CRC1039 (A03 to IT; and Z01 core) and the individual DFG research fund TEG322-11/1. Acknowledgements The authors would like to thank Sandra Trautman and Yannick Schreiber for technical support in performing the LC-MS/MS experiments. Author contributions CF did the behavioral analyses, tissue collection and initial analysis of behavioral data. DT and RG analyzed sphingolipids, eCB and LPA and edited the methods description, IT initiated the study, obtained funding and ethical approval, collected, and analyzed data, made the figures, and wrote the manuscript. All authors participated in writing or editing parts of the manuscript and agree with the last version of the manuscript. Competing interests The authors declare that they have no financial or other competing interests. The funding institution had no role in data acquisition, analysis, or decision to publish the results. Animal Ethical Approval Animal studies are reported and were conducted in compliance with the ARRIVE guidelines [61]. The experiments were approved by the local Ethics Committee for Animal Research (Darmstadt, Germany V 54 - 19 c20/15 - FK1074) and adhered to the European guidelines and to those of GV-SOLAS for animal welfare in science. Data availability statement Data are included in the manuscript or supplement. 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Supplementary Files CUMSschedule.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Aug, 2024 Read the published version in Pflügers Archiv - European Journal of Physiology → Version 1 posted Reviewers agreed at journal 19 May, 2024 Reviewers invited by journal 17 May, 2024 Editor assigned by journal 14 May, 2024 Submission checks completed at journal 14 May, 2024 First submitted to journal 12 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4408665","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":305618237,"identity":"ec278711-685e-40b5-a725-5d33518ee130","order_by":0,"name":"Caroline Fischer","email":"","orcid":"","institution":"Goethe-University Frankfurt","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Fischer","suffix":""},{"id":305618238,"identity":"83b3187d-6f58-4532-add8-3185d1a8181e","order_by":1,"name":"Dominique Thomas","email":"","orcid":"","institution":"Goethe-University Frankfurt","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dominique","middleName":"","lastName":"Thomas","suffix":""},{"id":305618239,"identity":"090ad582-3a05-44fe-801a-5317281825b3","order_by":2,"name":"Robert Gurke","email":"","orcid":"","institution":"Goethe-University Frankfurt","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Gurke","suffix":""},{"id":305618240,"identity":"864c8275-efd0-4eb6-a0ee-10dcf728401d","order_by":3,"name":"Irmgard Tegeder","email":"data:image/png;base64,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","orcid":"","institution":"Goethe-University Frankfurt","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Irmgard","middleName":"","lastName":"Tegeder","suffix":""}],"badges":[],"createdAt":"2024-05-12 13:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4408665/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4408665/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00424-024-03012-0","type":"published","date":"2024-08-23T15:57:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56990557,"identity":"79138ca2-455e-4b80-a329-6ea0fbf5fe09","added_by":"auto","created_at":"2024-05-23 06:15:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":570306,"visible":true,"origin":"","legend":"\u003cp\u003eBehavior in a chronic unpredictable mild stress (CUMS) model of chronic environmental stress in mice. CUMS mice (n = 11) were exposed to mild daily stressors including cage switch, itch, cold exposure, predator odor, headache, no bedding, wet bedding, lights on overnight, no food overnight, no water overnight, restraint, cage shaking, tilted cage or ultrasound noise for 6 weeks. Control mice (n = 10) were kept in neighboring cages without stress. Behavioral tests were done in the final CUMS week or at the end of the CUMS protocol.\u003c/p\u003e\n\u003cp\u003eA: Body weight before, during and at the end of the CUMS protocol.\u003c/p\u003e\n\u003cp\u003eB: Numbers of buried marbles in the Marble Burying Test (MBT, 12 marbles provided) within 30 min during and at the end of the CUMS protocol.\u003c/p\u003e\n\u003cp\u003eC: Immobility time in the Tail Suspension Tests (TST) in three trials in the last three days of the CUMS protocol, one trial per day.\u003c/p\u003e\n\u003cp\u003eD-F: Path lengths (D) and times spent (E) in open and closed arms and the center compartment in Elevated Plus Maze (EPM) test, and total distances traveled during the EPM observation of 10 min (F).\u003c/p\u003e\n\u003cp\u003eG: Latency to first licking from a standard bottle providing sweet water (2% sucrose in water) in the home cage and in an unfamiliar cage.\u003c/p\u003e\n\u003cp\u003eH: Water and sucrose-water intake (ml) in a 2-choice sucrose preference test (SPT) in home cages.\u003c/p\u003e\n\u003cp\u003eThe boxes show the interquartile range, the line is the median, whiskers show minimum to maximum, scatters represent mice. Data were submitted to 2-way ANOVA (group X time, or group x EPM arm etc.) and subsequent posthoc t-tests comparing CUMS versus control. Total EPM distances (F) were compared with 2-sided unpaired Student's t-test. The asterisks show significant differences between CUMS and control (* P \u0026lt; 0.05, ** P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/5f5d861f09cbda94b0aab088.png"},{"id":56990560,"identity":"2c821231-2beb-42bf-bbcf-8075e35be7ad","added_by":"auto","created_at":"2024-05-23 06:15:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":948136,"visible":true,"origin":"","legend":"\u003cp\u003eCircular map of CUMS evoked changes of plasma lipids. Plasma concentrations were obtained by targeted UPLC-MS/MS, and data are revealed as percentages of the median concentration of the respective lipid, which was set to 100%. The graphs show the group averages. Abbreviations: AEA, anandamide; OEA, oleoylethanolamide; PEA, palmitoylethanolamide; AG, arachidonoylglycerol; Cer, ceramides; GlcCer, glucosylceramides; LPA, lysophosphatidic acid; SPH, sphingoid base (sphingosine d18:1, sphinganine d18:0); S1P, sphingosine/anine-1-phosphate\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/bf064bc3420c79ba49e16895.png"},{"id":56990997,"identity":"2f7cc3f9-00e3-4baa-9089-1c34265f261c","added_by":"auto","created_at":"2024-05-23 06:23:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":346883,"visible":true,"origin":"","legend":"\u003cp\u003eCanonical linear discrimination analysis of brain lipids in CUMS and control mice. The analysis was set to discriminate brain regions using concentrations of 14 lipid species (endocannabinoids, sphingoid bases, ceramides and hexosylceramides) of CUMS mice (n = 11) which was used as the training set and applied to controls. The bubble plot shows the scores of the canonical discrimination variables 1 versus 2. Brain sites are color coded, closed symbols show CUMS mice, the bubbles sizes represent mouse age. Abbreviations: Cb, cerebellum; HC, hippocampus; MB, midbrain; PFC, prefrontal cortex; Th, thalamus; STR, striatum.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/2e893b18cc1390835431a02f.png"},{"id":56990998,"identity":"a5379759-e2aa-4e76-be03-8fb57c2758c1","added_by":"auto","created_at":"2024-05-23 06:23:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1101788,"visible":true,"origin":"","legend":"\u003cp\u003eCircular map of CUMS evoked changes of brain lipids at different brain regions. Tissue concentrations were obtained by targeted UPLC-MS/MS, and data are revealed as percentages of the median concentration of the respective lipid at the respective site, which was set to 100%. The graphs show the group averages. Abbreviations as in Figure 2.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/cee542c9e94af904fd09ede4.png"},{"id":56990565,"identity":"523d5e9b-e5f9-4c5a-818f-ba8c4f42e8c6","added_by":"auto","created_at":"2024-05-23 06:15:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":837457,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation plots showing associations of brain lipids across sites and behavior.\u003c/p\u003e\n\u003cp\u003eA: Correlation plots of control mice. B: Correlation plot of CUMS mice. The correlation is color coded as revealed in the scale bar. Red denotes high positive correlation, blue high negative correlation. In addition, the size of the dots shows correlation (positive or negative). Please note the strong differences of the S1P d18:1 and S1P d18:0 correlations, and of LacCer (lactosylceramides).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/d07727cb47ba5ae2afce761f.png"},{"id":56990567,"identity":"0d4d8352-7e7a-4294-b067-2e96bda0ff3d","added_by":"auto","created_at":"2024-05-23 06:15:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":746599,"visible":true,"origin":"","legend":"\u003cp\u003eEndocannabinoids at different brain sites and association of AEA with behavior of anxiety.\u003c/p\u003e\n\u003cp\u003eA: Box/scatter plots show endocannabinoid concentrations in different brain regions and OEA and PEA in plasma. The boxes show the interquartile range, the line is the median, whiskers show minimum to maximum, scatters represent mice. Data were submitted to 2-way ANOVA (group X site) and subsequent posthoc t-tests comparing CUMS versus control. The asterisks show significant differences between CUMS and control (* P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eB, C: Linear regression analysis of AEA concentrations in hippocampus (HC) with behavioral readouts of anxiety in the EPM (path in closed arm) and in the sucrose latency test in an unfamiliar cage. Table inserts show the regression parameters and P-values. The bubble size represents the time in closed arms (EPM, B) or the sucrose water consumption (drinking volumes) in C. The line shows the linear fit, the bands show the 90% confidence intervals.\u003c/p\u003e\n\u003cp\u003eAbbreviations: AEA, anandamide; OEA, oleoylethanolamide; PEA, palmitoylethanolamide; AG, arachidonoylglycerol; Cb, cerebellum; HC, hippocampus; MB, midbrain; PFC, prefrontal cortex; Th, thalamus; STR, striatum.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/a076f70942e39a5ace525109.png"},{"id":56990563,"identity":"f01de08b-5388-4440-bc83-10aa219e36f6","added_by":"auto","created_at":"2024-05-23 06:15:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":325739,"visible":true,"origin":"","legend":"\u003cp\u003eSphingosine, sphinganine and S1P at different brain sites.\u003c/p\u003e\n\u003cp\u003eBox/scatter plots show concentrations of sphingoid bases (sphingosine and sphinganine) and phosphorylated sphingoid bases (S1P d18:1, S1P d18:0) in different brain regions and S1P in plasma. The boxes show the interquartile range, the line is the median, whiskers show minimum to maximum, scatters represent mice. Data were submitted to 2-way ANOVA (group X site) and subsequent posthoc t-tests comparing CUMS versus control. The asterisks show significant differences between CUMS and control (* P \u0026lt; 0.05, **\u0026lt;0.01, ***\u0026lt;0.001, ****\u0026lt;0.0001). Brain site abbreviations as in Figure 6.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/21d06facbf2dedb54399dd72.png"},{"id":56990566,"identity":"c36becfa-71df-4502-90fa-2bf05fc775d1","added_by":"auto","created_at":"2024-05-23 06:15:24","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":647313,"visible":true,"origin":"","legend":"\u003cp\u003eAssociations of S1P in thalamus with behavioral readouts of stress resilience\u003c/p\u003e\n\u003cp\u003eA, B: Linear regression analysis of S1P d18:0 (sphinganine-1-phosphate) concentrations in midbrain with EPM path in closed arm (but not with time in the closed arm, bubble size) and sweet water consumption (n.s.) There was no association with sucrose-water latency (bubble size).\u003c/p\u003e\n\u003cp\u003eC, D: Linear regression analysis of S1P d18:1 (sphingosine-1-phosphate) concentrations in thalamus with the number of buried marbles in the MBT and the immobility time in the tail suspension test (TST) and. The higher S1P, the more marbles were buried, showing strong defensive efforts. In agreement, the higher the S1P levels, the lower was the immobility time, which is the inverse of the struggling time. Strong struggling indicates high defensive efforts.\u003c/p\u003e\n\u003cp\u003eTable inserts show the regression parameters and P-values. The line shows the linear fit, the bands show the 90% confidence intervals.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/0650961d6344234e24b6f70f.png"},{"id":56990561,"identity":"2668878e-ab41-4552-baf6-3f0c88d3d107","added_by":"auto","created_at":"2024-05-23 06:15:23","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":554208,"visible":true,"origin":"","legend":"\u003cp\u003eCeramides and hexosylceramides at different brain sites and in plasma\u003c/p\u003e\n\u003cp\u003eA, B: Box/scatter plots show concentrations of ceramides (A) and hexosylceramides (B) in different brain regions and in plasma. The boxes show the interquartile range, the line is the median, whiskers show minimum to maximum, scatters represent mice. Data were submitted to 2-way ANOVA (group X site) and subsequent posthoc t-tests comparing CUMS versus control. The asterisks show significant differences between CUMS and control (* P \u0026lt; 0.05, **\u0026lt;0.01, ***\u0026lt;0.001). Brain site abbreviations as in Figure 6. Lipids: Cer, Ceramides; LacCer, lactosylceramides; GlcCer, glucosylceramides\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/9f5920df8989a5f7bfc25fa7.png"},{"id":63300252,"identity":"83a819e0-b78d-4076-8388-2ee99ea0a524","added_by":"auto","created_at":"2024-08-26 16:13:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5388557,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/d0a6803f-860f-404a-81e2-5aa031dc7741.pdf"},{"id":56990559,"identity":"f189fdc8-9721-41d0-af4c-878f8333102b","added_by":"auto","created_at":"2024-05-23 06:15:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":388416,"visible":true,"origin":"","legend":"","description":"","filename":"CUMSschedule.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4408665/v1/6fcd2e37e5d011089f8f45f3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Brain region specific regulation of anandamide (down) and sphingosine-1- phosphate (up) in association with anxiety (AEA) and resilience (S1P) in a mouse model of chronic unpredictable mild stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStress exposure is normal in daily life, but it is also a predisposing risk factor for metabolic, cardiovascular, and mental disease such as anxiety and depression [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Stressors do not influence all individuals equally. Some are vulnerable while others are more resilient [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The adaptations of the brain to acute and short-lasting stressful conditions involves neuronal systems that also process rewarding stimuli [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Studies indicate that the ability to cope with acute or chronic stress relies in part on the neurotransmitter, dopamine, mostly considered as a signal of reward or stress relief [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, dopamine is also involved in aversive motivation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Acute stress increases extracellular dopamine levels in the mesolimbic mesocortical dopamine system, particularly nucleus accumbens and prefrontal cortex (PFC) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], but chronic or intense stressors, particularly prolonged, repetitive, unpredictable and unavoidable stress rather inhibits dopamine release or dopamine receptor responsiveness [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In rodents, chronic restraint stress, chronic social defeat stress, and chronic unpredictable mild stress (CUMS) are associated with depressive-like behaviors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and are linked to suppressed midbrain dopaminergic neuronal activity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere is some evidence from previous studies that microbiome derived lipids [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and endogenous bioactive lipids including prostaglandins, sphingolipids and endocannabinoids contribute to the mal-adaptations to chronic stress at the molecular level [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and in turn, are regulated by chronic or acute stress [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Ceramides are believed to aggravate or maintain aversiveness [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and have been suggested as putative targets for antidepressants [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] whereas endocannabinoids (eCBs) protect against psychological sequelae of chronic stress [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], likely as positive indirect modulators of the dopamine system. DA neurons in the midbrain receive excitatory glutamatergic and inhibitory GABAergic input [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and both are under inhibitory control via presynaptic cannabinoid receptors [\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Functions of sphingoid-base sphingolipids are unclear and mostly based on studies with FTY720 (fingolimod [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]), which acts as a sphingosine-1-phosphate (S1P) receptor agonist after phosphorylation but leads to receptor downregulation. One study revealed that S1P-dependent stress resilience required signaling through S1P receptor 3 (\u003cem\u003eS1PR3\u003c/em\u003e) in medial prefrontal cortex [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], but alternative effects such as inhibition of microglial inflammasome activation were also suggested [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe evidence of a mutual endocannabinoid-mediated stress-lipid signaling arises form preclinical studies with genetic ablation or pharmacologic antagonism of the cannabinoid type 1 receptor (\u003cem\u003eCnr1\u003c/em\u003e, CB1) [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], or from clinical experience with the CB1-antagonist, rimonabant [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Knockout of CB1 results in exaggerated neuroendocrine and behavioral responses to acute stress including anxiety, reward sensitivity, pain, morphologic changes in the amygdala, and hippocampal synaptic plasticity [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44 CR45 CR46\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Under acute stress, endocannabinoids limit the magnitude of the stress response and facilitate recovery after cessation of stress exposure [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, under chronic or severe stress, the endocannabinoid system appears to \u0026ldquo;collapse\u0026rdquo; in the sense that CB1 receptors are downregulated or dysfunctional or that endocannabinoids are missing [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Hence, the ability to modulate the synaptic release of neurotransmitters such as glutamate and gamma-aminobutyric acid is lost [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Consequently, inhibitors of endocannabinoid breakdown attenuated stress evoked behavioral manifestations of depression or anxiety [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In humans, posttraumatic stress disorder or major depressive disorder is associated with reductions of the circulating levels of endocannabinoids [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], and a significant proportion of individuals using the CB1 antagonist rimonabant for weight loss developed indices of anxiety and depression and suicide and finally, withdrawal of the drug [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on these studies we hypothesized that chronic stress leads to brain-region dependent changes of endocannabinoids, which in turn would increase the suffering from chronic stress. The vicious cycle may be fixated by genetic or epigenetic modifications and further profound changes of connected or independent lipid signaling paths that as a whole determine the behavioral outcome and susceptibility to mental disease on one side and gain of resilience or even reward upon stress relief on the other side. To address this hypothesis, we analyzed lipids of emotion-relevant classes (endocannabinoids, ceramides, hexosylceramides and sphingoid bases) in seven brain regions and plasma in association with behavior in a model of chronic unpredictable mild stress in mice.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMice\u003c/p\u003e \u003cp\u003eAnimal studies are reported and were conducted in compliance with the ARRIVE guidelines [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The experiments were approved by the local Ethics Committee for Animal Research (Darmstadt, Germany V 54\u0026thinsp;\u0026minus;\u0026thinsp;19 c20/15 FK1074) and adhered to the European guidelines and to those of GV-SOLAS for animal welfare in science. We used female mice which were raised in the local breeding facility. Matched pairs according to body weight and age were submitted to chronic unpredictable mild stress (CUMS; n\u0026thinsp;=\u0026thinsp;11) and control groups (n\u0026thinsp;=\u0026thinsp;10). Group assignment was blinded. The ages at the start ranged from 6\u0026ndash;15 weeks (average 9.5 weeks) in both groups. The average weight at onset was 20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 g (CUMS) and 20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 g (control). Mice were housed in pairs (except one cage with n\u0026thinsp;=\u0026thinsp;3) and kept in a controlled environment (12-hour dark/light cycle, 23\u0026deg;C, 55% humidity) with food and water ad libitum. Animal numbers were estimated by power analysis using GPower [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The probability of type-1 error was set to α\u0026thinsp;=\u0026thinsp;0.05, and type-2 error was set at β\u0026thinsp;=\u0026thinsp;0.2.\u003c/p\u003e \u003cp\u003eChronic unpredictable mild stress (CUMS)\u003c/p\u003e \u003cp\u003eTo evoked mild stress mice are exposed daily to different types of mild stressors, such as temporary isolation or crowded housing, tilted home cages, wet bedding, predator odor, cold exposure, restraint, itch, no food, no water or disrupted dark-light cycle [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The time of each stressor varies from 0.5-4 h per day or overnight, and the protocol can last for 3\u0026ndash;12 weeks. The detailed protocol used in this study is provided as Supplementary timetable. To integrate behavioral tests of control mice the stress protocol lasted for 6 weeks with a short break of 6 days without stressors. Behavioral tests of CUMS mice were integrated in the last stress exposure days. The stressors included cage switch, itch, cold exposure, TMT odor, headache (nitroglycerin i.p.), no bedding, wet bedding, lights on overnight, no food overnight, no water overnight, restraint, cage shaking, tilted cage, ultrasound noise. The behavior tests using tail suspension and elevated plus maze per se are also considered as stressors.\u003c/p\u003e \u003cp\u003eElevated Plus Maze (EPM)\u003c/p\u003e \u003cp\u003eThe EPM takes advantage of the physiologic self-protective hiding of mice in the dark versus their curiosity. The test is considered to measure anxiety-like behavior. The standard EPM maze was configured with two orthogonally arranged closed arms (25L \u0026times; 5W \u0026times; 15H cm) and two open arms (25L \u0026times; 5W \u0026times; 0.3H cm) connected by a central open square (5 \u0026times; 5 cm), and it was elevated 60 cm above the floor and illuminated from above. The floor and walls were made of grey PVC. The maze was placed in a quiet enclosure of the test room and mice were habituated before start. At test start, mice were placed individually into the center platform facing an open arm and were allowed to move freely for 10 min [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The behavior was video recorded with a camera mounted above the maze. VideoMot2 (TSE Systems GmbH, Bad Homburg, Germany) was used for automatic tracking and analysis of arm entries, times in closed/open and distances. The anxiety index was calculated for as AI = (time in closed/total time in arms) \u0026ndash; (time in open/total time in arms).\u003c/p\u003e \u003cp\u003eTail Suspension Test (TST)\u003c/p\u003e \u003cp\u003eIn the TST mice are suspended by the tail, which elicits defensive struggling and immobile hanging. The latter is often interpreted as resignation or depression resulting from an unsolvable and aversive situation [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], and the time of immobility the primary readout. We used a self-constructed observation chamber which was divided into five compartments, separated by dark grey spacers, each compartment with hook 30 cm above the floor. A widefield camera was mounted in front on a tripod to capture five mice simultaneously. The tail was fixed with adhesive tape to the hook, and each test lasted 10 min [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. The video recordings were analyzed posthoc by using a computer key to measure the time and was done without knowledge of the group assignment.\u003c/p\u003e \u003cp\u003eSucrose preference and latency\u003c/p\u003e \u003cp\u003eMice are highly addicted to sweet water. Compared with daily tap water volumes of about 4 ml, they may consume up to 16 ml sweet water (2% sucrose). High sucrose preference may be interpreted as consolation, or compulsiveness/addiction, depending on the extent and choices. To measure sucrose preference, a 2-choice test was used. Mice were housed in pairs per cage and habituated for 3 days by providing bottles of tap water on the right and left side of the cage. One bottle was then replaced with 5% sucrose in tap water, randomly on the right or left side. The volume intake was assessed by daily weighing the bottles. The latency to first licking of sucrose-water after an overnight water restriction was assessed in the home cage and in a new cage with new bedding. Mice were tested individually. The latency was assessed by observation with a stopwatch.\u003c/p\u003e \u003cp\u003eMarble burying\u003c/p\u003e \u003cp\u003eThe marble burying test has been used to assess stress or novelty evoked anxiety (defensive burying or neophobia burying) [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] or assessment of repetitive and compulsive behavior [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The test takes advantage of the normal spontaneous burying and digging behavior. Increased marble burying under stress reflects an inherent defensive response aimed at protecting from harmful objects. The behavioral meaning of marble burying is however debatable [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. For the MBT, 12 neutral 0.5-inch glass marbles were arrayed on the surface of clean, thick 7 cm bedding. The number of buried marbles (covered at least \u0026frac34; with bedding) was counted at 5 min, 10 min, 20 min and at the end of a 30 min observation period. The MBT was done during and after completion of the CUMS protocol. The 30 min end point of \"MBT-during-CUMS\" was used for lipid association analyses. The investigator was unaware of the group assignment.\u003c/p\u003e \u003cp\u003eTissue and blood sampling\u003c/p\u003e \u003cp\u003eMice were euthanized by CO2 in a chamber with adjustable stepwise increasing flow. Blood samples were collected in 500 \u0026micro;l K3-EDTA microtubes (Microvette Sarstedt) after cessation of respiration via cardiac puncture with a 27G needle attached to a 1 ml syringe. The samples were immediately centrifugated in a mini- tabletop centrifuge at 2000 \u003cem\u003eg\u003c/em\u003e for 3 min, plasma transferred into microtubes using a 100 \u0026micro;l pipette and directly frozen in liquid nitrogen and kept at -80\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eThe brain war rapidly removed, olfactory bulb removed (discarded), cerebellum collected, weighed and frozen in liquid nitrogen, then brain cut sagittal, the halves unfolded, and then regions collected from rostral to dorsal: orbitofrontal and dorsal prefrontal cortex, striatum including nucleus accumbens, hippocampus, thalamus, hypothalamus, and midbrain. All samples were weighed on a precision scale and directly frozen in liquid nitrogen and kept at -80\u0026deg;C until analysis. Lipid concentrations are normalized on mg of tissue. Orbitofrontal and dorsal prefrontal cortex samples were analyzed separately, but concentrations were then averaged to get one PFC-value per mouse to reduce brain sites and gain power.\u003c/p\u003e \u003cp\u003eAnalysis of lipid signaling molecules\u003c/p\u003e \u003cp\u003eBioactive lipids including sphingoid bases and ceramides, lysophosphatidic acids (plasma only), and endocannabinoids (eCBs) were analyzed in plasma and in brain tissue homogenates using liquid-liquid-extraction (LLE) followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) as described in detail in a previous study [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, brain tissue samples were homogenized in ethanol:water (1:3, v/v) using a Mixer Mill MM400 (Retsch, Haan, Germany). Afterwards, brain tissue homogenates as well as plasma samples were extracted using an LLE protocol as described in detail in the previous publication. Sphingolipids were separated using an Agilent 1200 HPLC system equipped with a Zorbax C18 Eclipse Plus UHPLC column (50 \u0026times; 2.1 mm, 1.8 \u0026micro;m, Agilent technologies, Waldbronn, Germany). The analysis of LPAs (plasma only) was done on the same HPLC system using a Luna C18 column (50 \u0026times; 2.0 mm, 5 \u0026micro;m, Phenomenex, Aschaffenburg, Germany). For the chromatographic separation of endocannabinoids an Agilent 1290 Infinity I UHPLC system equipped with an Acquity UPLC BEH C18 UPLC column (100 \u0026times; 2.1 mm, 1.7 \u0026micro;m, Waters, Eschborn, Germany) was used. The quantification of all analytes was performed using a hybrid triple quadrupole-ion trap mass spectrometer QTRAP 5500 or 6500+ (Sciex, Darmstadt, Germany) equipped with a Turbo-V-source operating in positive ESI mode for sphingolipids and endocannabinoids and in negative ESI mode for LPAs.\u003c/p\u003e \u003cp\u003eQuality control samples of three different concentration levels (low, middle, high) were run as initial and final samples of each run. For all analytes, the concentrations of the calibration standards, quality controls and samples were evaluated by Analyst software 1.6 and MultiQuant software 3.0 (Sciex, Darmstadt Germany) using the internal standard method (isotope-dilution mass spectrometry). Calibration curves were calculated by linear or quadratic regression with 1/x weighting or 1/x2 weighting.\u003c/p\u003e \u003cp\u003eTo assess putative biases caused by sample sequence, concentrations were plotted versus analysis number. For ethanolamide endocannabinoids (AEA, OEA, PEA) linear regression analysis revealed a shallow but significant linear raise (significantly different from zero) with the sample number. The concentrations were therefore adjusted according to analysis sequence number and the slope of the regression line. There was no effect of sample sequence for any of the other analytes.\u003c/p\u003e \u003cp\u003eStatistics\u003c/p\u003e \u003cp\u003eLipid concentrations are presented as scatter plots with mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) or box-scatter plots, where the box is the interquartile range and the whiskers show minimum to maximum, or the 95% confidence interval (CI), specified in the figure legend. Data were analyzed with SPSS 29, Origin Pro 2024 and GraphPad Prism 9.0. Principal component analysis (PCA) and partial least square analysis (PLS) were used to reduce dimensionality and identify the factors which contributed most to the difference between treatment groups and brain sites. In addition, linear canonical discriminant analysis (DA) was used to assess the predictability of group membership based on DA scores. DA was performed without and with bootstrapping, the latter using a stratified random sampling approach considering gender and age, and 100 iterations.\u003c/p\u003e \u003cp\u003eLipid concentrations and behavioral readouts were compared between groups using analyses of variance (two-way ANOVA for brain site X treatment (CUMS or control), or t-tests according to the data subgroup structure and distribution. In case of significant results of ANOVAs, treatment groups were compared per brain site using t-tests comparing CUMS versus control. P-values were adjusted according to Šid\u0026aacute;k for multiple comparisons or were not adjusted if only two groups were compared. For behavioral time course data, 2-way ANOVA for repeated measurements \"time\" X \"group\" was used. The alpha level was set at 0.05 for all comparisons and asterisks in the figures refer to adjusted P-values.\u003c/p\u003e \u003cp\u003eFor cluster analyses and polar plots, lipid concentrations were normalized to the median of all samples of the respective lipid to allow for a combined analysis and presentation. For correlation plots, lipids and behavioral readouts were scaled and correlations are coded by color (blue negative correlation, red positive correlation) and bubble size according to the adjusted R-square. Further analyses consisted in multiple regression analyses to reveal associations between behavioral readouts and lipids that were different between CUMS and control mice at one or more brain sites. The regression analysis only included the CUMS groups based on the hypothesis that an increase or decrease of the respective lipid would be associated with a change of behavior rather than that the lipid concentration as such would predict a behavior. There was no association of behavior for any lipid in the control group.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCUMS evoked mild weight loss and anxiety\u003c/p\u003e \u003cp\u003eAs expected CUMS was associated with mild effects on health and behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-H) manifesting in a mild reduction of the body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), increase of plasma ceramides (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and decrease of the time spent in open arms of the EPM (1D, E), which may be interpreted as anxiety-like behavior. In addition, the latency to first licks of sweet water was increased in an unfamiliar novel cage as compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), but sucrose intake was not different (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). The behavior agrees with anxiety-like behavior (\"neophobia\") rather than \"depression\". There was no difference in marble burying behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and total distance moved in maze tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In the TST, immobility time was reduced in the first trial showing that CUMS mice spent more time with struggling but was not consistently maintained upon repeated testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003ePlasma concentrations of endocannabinoids and sphingolipids were analyzed to assess the systemic metabolic impact of CUMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Ceramides were increased, particularly Cer d18:1/16:0 and Cer d18:1/22:0 which agrees with the expectation that stress disrupts metabolic homeostasis. High plasma concentrations of ceramides have been associated with obesity, diabetes [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] and mental disease (major depression, bipolar disorder) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, endocannabinoids, OEA and PEA were increased, likely elicited by the loss of body weight during CUMS and favoring rapid body weight regain after CUMS (sample were taking after BW recovery) [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBrain regions have site-specific lipid patterns\u003c/p\u003e \u003cp\u003eLipid species of four classes (4x endocannabinoids, 4x sphingoid bases, 4x ceramides, 5x hexosylceramides) of seven brain sites were submitted to linear canonical discrimination analysis to reveal brain site specific patterns and effects of CUMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Scatter plots of the discriminant scores (canonical variable 1 versus 2) show that and lipid patterns of midbrain (MB) and thalamus (Th) are similar, that the cortical patterns of PFC and hippocampus are closely related, and striatum is highly variable in between. The cerebellum is unique in its lipid patterns. The discrimination task was to separate sites and CUMS levels were used for learning and applied to controls. The analysis shows that the patterns apply to both groups in agreement with the expectation of the model, which was meant to cause mild stress but no profound disruption.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effects CUMS however do reveal at the level of individual lipid species and are site-specific. In the first set of analyses presented as circular plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) data were transformed to percentages versus overall median to visualize the effect of CUMS. The forebrain to midbrain (top to bottom) the CUMS group impresses with reduced anandamide and lactosylceramides in PFC, increase of sphingoid bases and ceramides in striatum and a further increase of sphingoid bases (but not ceramides) in thalamus and midbrain. Across brain regions, S1P d18:1 (sphingosine-1-phosphate) and S1P d18:0 (sphinganine-1-phosphate) are higher in the CUMS group.\u003c/p\u003e \u003cp\u003eCorrelation of lipids across brain regions with behavioral readouts\u003c/p\u003e \u003cp\u003eLipid levels across brain regions (i.e., the average of all sites for each mouse), body weights and behavioral readouts were submitted to correlation analyses and plotted as correlation plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) to reveal if and how brain lipids or behavioral features were associated with each other. The analysis was done for control and CUMS separately to compare patterns. The CUMS correlation map has more positive (red) and negative (blue) correlations. Again, S1P and LacCer show the strongest differences. In controls, S1P is negatively correlated with ceramides, which is lost or inverted in CUMS. In CUMS there are strong negative correlations of AEA with behavior which is not evident in controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCorrelation maps, circular lipid plots and behavior suggested two hypotheses (i) anandamide (AEA) deficiency in hippocampus and cortex evoked by CUMS is associated with anxiety and (ii) S1P in subcortical structures is associated with defensive and combative depression-averting behavior. To address the hypothesis individual site-specific lipid concentrations were analyzed in detail in associations with behavioral features using multiple regression analyses (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCUMS-evoked low AEA in cortex associated with anxiety\u003c/p\u003e \u003cp\u003eAEA was significantly reduced in the PFC in CUMS mice versus controls, and OEA was increased in the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). As revealed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, OEA and PEA were increased in plasma of CUMS versus control mice. AEA is a canonical cannabinoid receptor ligand, and therefore associations with behavior were primarily assessed for AEA. The hippocampus was chosen as primary site (although n.s.) because the HC concentrations in CUMS mice showed a broad range suggesting that HC-AEA may reflect the individual susceptibility to CUMS evoked behavior. Linear regression analyses revealed that low AEA in hippocampus (or PFC not shown) was significantly associated with long distance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) (or long time, not shown) in the closed arms in EPM. The lower AEA, the more time was spent in the closed arm, suggesting that low AEA increased anxiety or reduced curiosity. Low AEA in the hippocampus was also associated with a long sucrose latency in an unfamiliar cage, which however did not reach statistical significance (P 0.062).\u003c/p\u003e \u003cp\u003eCUMS-evoked high S1P in thalamus and midbrain associated with TST struggling and MBT\u003c/p\u003e \u003cp\u003eS1P d18:1 (sphingosine-1-phosphate) and S1P d18:0 (sphinganine-1-phosphate) were increased in midbrain and thalamus in CUMS mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In addition, non-phosphorylated sphinganine was increased in hippocampus, PFC, and cerebellum in CUMS versus control. Linear regression analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e) show that high S1P d18:0 (sphinganine-1-phosphate) is associated with long paths in the closed EPM arms (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). However, there was no association with time spent in closed arms (not shown, linear adj R-square \u0026minus;\u0026thinsp;0.111; ANOVA P-value 0.958) showing that mice with high S1P d18:0 in midbrain were more active within the closed arms. There was no association of S1P d18:0 with sucrose latency (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). S1P d18:1 in the thalamus (or across regions, not shown) was associated with a high number of buried marbles in the MBT (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) and high struggling time in TST (which is the inverse of the immobility time) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eAlthough previous studies in mice suggest that ceramides are particularly important for stress-evoked depression like behavior and plasma ceramides agree with the expected stress evoked increase, there were no consistent between group differences, and no significant association between plasma and across-brain ceramides levels. Notably, ceramides were high in striatum in both groups compared with other brain regions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study shows that CUMS in mice leads to mostly subtle site-specific adjustments of endocannabinoids, particularly a loss of AEA in the PFC and increase of sphingolipids, particularly S1P in midbrain and thalamus. Low AEA in PFC and hippocampus are associated with anxiety-like behavior i.e., preference of dark zone in EPM and long sucrose-latency in an unfamiliar cage. These results agree well with previous studies of chronic stress in mice where AEA was found reduced in the brain, albeit not specifically in cortical structures but throughout studied brain regions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Other studies using electrophysiology and CB1 receptor knockout have suggested that specifically the endocannabinoid systems of the hippocampus and amygdala have a key role in anxiety extinction [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e] and stress relief [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Human imaging studies using cannabinoid radioligands reveal increases of free CB1 binding capacity in stress associated diseases such as posttraumatic stress disorder suggesting a relative deficiency of endocannabinoids at affected sites [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Brain samples in our study were collected directly at the end of the CUMS protocol, hence not allowing mice to learn that the stress period was finished. Therefore, we assume that the brain was captured in a state of heightened alertness, unease and fear for the next stressor and not in a state of stress relief, which is expected to switch the brain into reward-mode [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] associated with an increase of endocannabinoids and enhancement of dopamine release from crucial reward sites of the mesolimbic system [\u003cspan additionalcitationids=\"CR82 CR83\" citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. The repeated experience of stress relief was shown to increase stress resilience and prevent depressive symptoms in mice [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResilience in mice is mostly inferred from non-occurrence of stress-evoked fear i.e., as a negative readout, and the meaning of specific behaviors is context-sensitive and complex. In the present study we interpreted the struggling behavior in the TST and the marble burying behavior in the MBT as positive readouts of active defensive behavior and hence resilience [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e], as opposing to depression that would manifest as immobility, low locomotion, reduced feeding or reduced nest building [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. This interpretation is supported by studies which use the immobility time in TST or small numbers of buried marbles as indicators of depression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR88\" citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. The MBT is particularly controversial. Digging and burying of noxious, harmless, or rewarding objects is part of normal food searching and nest building behavior, and it is expressed in home cages without stress and under anxiogenic circumstances for example imposed by exposure to noxious objects or predator odor [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Defensive burying has been defined as the process of moving bedding material to cover harmful stimuli such as sources of electrical foot shock, and it is used as a measure of aversive anxiety [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. In addition, burying of novel objects under stressful conditions is considered as neophobia but mostly does not well agree with other readouts of novelty-evoked fear [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], and excessive marble burying was suggested to reveal nonfunctional repetitive behavior analogous to behavioral symptoms of obsessive\u0026ndash;compulsive disorder [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e], impulsivity, autism, and dementia, however with low predictive value for therapeutics of such disorders [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. Hence, as a stand-alone test, MBT offers alternative interpretations. In the present study, the numbers of buried marbles in CUMS mice were more variable but not significantly different from those of control mice but were positively correlated with the struggling time in the TST, which is the inverse of the immobility time, and with S1P d18:1 and S1P d18:0 levels in the thalamus and across brain regions. It is not much known about putative functions of S1P in the brain for adjustments of the brain towards chronic stress and coping with chronic stress. However, one study revealed that fingolimod increased stress resilience (i.e., prevented stress-evoked depression-like behavior) in a model of chronic unpredictable stress via activation of S1PR3 in the medial prefrontal cortex [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Once phosphorylated, fingolimod is an S1P receptor agonist [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. However, it is mostly recognized for opposite net effects, i.e., reduction of S1P signaling resulting from receptor downregulation. The latter effect prevents T-cells from egress of secondary lymphoid organs, hence explaining its therapeutic efficacy in autoimmune-mediated diseases, particularly preventing relapse in multiple sclerosis [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. Major depression, schizophrenia, PTSD, and other psychiatric diseases are believed to be contributed or sustained by inadequate immune activation [\u003cspan additionalcitationids=\"CR98 CR99\" citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]. Hence, many studies assessed the efficacy of fingolimod in experimental models of such psychiatric diseases mostly with some therapeutic benefit for fingolimod treated mice [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e, \u003cspan additionalcitationids=\"CR102 CR103\" citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e], which was however not confirmed in clinical studies [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. Considering differences in treatment schedules in mice and psychiatric patients, it may be suggested that long-term fingolimod leads to receptor downregulation in the brain similar to its regulation in immune cells and hence, interferes with resilience strengthening effects of endogenous brain S1P under stress conditions. Indeed, depression is frequent in MS patients receiving fingolimod, but switching to fingolimod from other disease modifying drugs was reported to reduce depression at least temporarily [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]. Although chronic stress evoked mental health issues are believed to be contributed by immune activation, we did not observe an increase of plasma S1P in CUMS mice. Instead, plasma lipids revealed an increase of ceramides, particularly Cer d18:1/22:0 which are increased in patients with major depression and bipolar disorder, again particularly Cer d18:1/22:0 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. Ceramides were not associated with body weight presumably because time points of blood sampling and weight loss during CUMS did not match. Blood was obtained at the end of the CUMS protocol when the body weight was already restored. Nevertheless, alterations of plasma ceramides well agree with the concept of stress-evoked unfavorable metabolic effects which however did not correlate with behavioral readouts of \"mental\" health in our mice.\u003c/p\u003e \u003cp\u003eIn summary, we show that CUMS in mice resulted in low AEA levels in cortical brain regions in correlation with anxiety-like behavior, and high S1P levels in midbrain and thalamus in correlation with defensive behavior. The results agree with previous studies of brain anandamide under stress and strengthen the idea that cannabinoids might be useful in certain cases of PTSD [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. S1P results are novel. They agree with strengthening of resilience with short-term fingolimod treatment in mice but suggest that sphingosine kinase inhibitors that are under investigation for cancer and fibrosis [\u003cspan additionalcitationids=\"CR111 CR112\" citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e] might affect mental health, so far not observed.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003earachidonoylglycerol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanandamide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCer\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCeramides\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLacCer\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLactosylceramides\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGlcCer\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglucosylceramides\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eeCB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eendocannabinoid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esphingolipids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS1P d18:1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esphingosine-1-phosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS1P d18:0\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSphinganine-1-Phosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCUMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echronic unpredictable mild stress\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEPM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eelevated plus maze\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esucrose preference tests\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etail suspension test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eScrLat\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSucrose licking latency\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eArticle dedication\u003c/h2\u003e\n\u003cp\u003eThe article is dedicated to our colleague, Andrea Huwiler, who recently passed away before the Special issue could be finalized.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe study was supported by the Deutsche Forschungsgemeinschaft (DFG) via the collaborative research center CRC1039 (A03 to IT; and Z01 core) and the individual DFG research fund TEG322-11/1.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank Sandra Trautman and Yannick Schreiber for technical support in performing the LC-MS/MS experiments.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eCF did the behavioral analyses, tissue collection and initial analysis of behavioral data. DT and RG analyzed sphingolipids, eCB and LPA and edited the methods description, IT initiated the study, obtained funding and ethical approval, collected, and analyzed data, made the figures, and wrote the manuscript. All authors participated in writing or editing parts of the manuscript and agree with the last version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no financial or other competing interests. The funding institution had no role in data acquisition, analysis, or decision to publish the results.\u003c/p\u003e\n\u003ch2\u003eAnimal Ethical Approval\u003c/h2\u003e\n\u003cp\u003eAnimal studies are reported and were conducted in compliance with the ARRIVE guidelines\u0026nbsp;[61].\u0026nbsp;The experiments were approved by the local Ethics Committee for Animal Research (Darmstadt, Germany V 54 - 19 c20/15 - FK1074) and adhered to the European guidelines and to those of GV-SOLAS for animal welfare in science.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eData availability statement\u003c/h2\u003e\n\u003cp\u003eData are included in the manuscript or supplement. Additional raw data are available upon reasonable scientific request from the corresponding author.\u003c/p\u003e\n\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDong TS, Gee GC, Beltran-Sanchez H, Wang M, Osadchiy V, Kilpatrick LA, Chen Z, Subramanyam V, Zhang Y, Guo Y, Labus JS, Naliboff B, Cole S, Zhang X, Mayer EA, Gupta A (2023) How Discrimination Gets Under the Skin: Biological Determinants of Discrimination Associated With Dysregulation of the Brain-Gut Microbiome System and Psychological Symptoms. 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Cell Signal 79:109881 DOI \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cellsig.2020.109881\u003c/span\u003e\u003cspan address=\"10.1016/j.cellsig.2020.109881\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sphingolipids, endocannabinoids, chronic unpredictable mild stress, ceramides, hippocampus, prefrontal cortex, thalamus, midbrain, anxiety, depression, resilience","lastPublishedDoi":"10.21203/rs.3.rs-4408665/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4408665/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic unpredictable and unavoidable stress is associated with mental health problems such as depression and anxiety, whereas cycles of stress and stress relief strengthen resilience. It has been suggested that increased breakdown of brain endocannabinoids (eCB) promotes a feeling of adversity. To assess the impact of stress on bioactive lipid homeostasis we analyzed eCB, sphingolipids and ceramides in seven brain regions and plasma in a mouse model of chronic unpredictable mild stress. CUMS was associated with low levels of anandamide in hippocampus and prefrontal cortex in association with indicators of anxiety (elevated plus maze). Oppositely, CUMS caused elevated levels of sphingosine-1-phosphate (S1P d18:1) and sphinganine-1-phosphate (S1P d18:0) in midbrain and thalamus, which was associated with readouts of increased stress resilience, i.e., marble burying and struggling in the tail suspension tests. In the periphery, elevated plasma levels of ceramides revealed similarities with human major depression and suggested unfavorable effects of stress on metabolism, but plasma lipids were not associated with body weight, sucrose consumption or behavioral features of depression or anxiety. The observed brain site specific lipid changes suggest that the forebrain succumbs to adverse stress effects while the midbrain takes up defensive adjustments.\u003c/p\u003e","manuscriptTitle":"Brain region specific regulation of anandamide (down) and sphingosine-1- phosphate (up) in association with anxiety (AEA) and resilience (S1P) in a mouse model of chronic unpredictable mild stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-23 06:15:19","doi":"10.21203/rs.3.rs-4408665/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"254833418248934613486262548663581482773","date":"2024-05-19T15:53:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-17T15:05:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-14T13:28:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-14T12:29:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pflügers Archiv - European Journal of Physiology","date":"2024-05-12T13:32:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6315f8ac-4bb9-4e4f-92db-1bd55a5626cd","owner":[],"postedDate":"May 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:03:01+00:00","versionOfRecord":{"articleIdentity":"rs-4408665","link":"https://doi.org/10.1007/s00424-024-03012-0","journal":{"identity":"pflugers-archiv-european-journal-of-physiology","isVorOnly":false,"title":"Pflügers Archiv - European Journal of Physiology"},"publishedOn":"2024-08-23 15:57:30","publishedOnDateReadable":"August 23rd, 2024"},"versionCreatedAt":"2024-05-23 06:15:19","video":"","vorDoi":"10.1007/s00424-024-03012-0","vorDoiUrl":"https://doi.org/10.1007/s00424-024-03012-0","workflowStages":[]},"version":"v1","identity":"rs-4408665","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4408665","identity":"rs-4408665","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}