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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Mol Psychiatry. 2015 Mar 10;21(1):80–88. doi: 10.1038/mp.2015.14

The impact of chronic stress on the rat brain lipidome

Tiago Gil Oliveira 1,2, Robin B Chan 3, Francisca Vaz Bravo 1,2, André Miranda 1,2, Rita Ribeiro Silva 1,2, Bowen Zhou 3, Fernanda Marques 1,2, Vítor Pinto 1,2, João José Cerqueira 1,2, Gilbert Di Paolo 3, Nuno Sousa 1,2,*
PMCID: PMC4565780  NIHMSID: NIHMS706514  PMID: 25754084

Abstract

Chronic stress is a major risk factor for several human disorders that affect modern societies. The brain is a key target of chronic stress. In fact, there is growing evidence indicating that exposure to stress affects learning and memory, decision making and emotional responses, and may even predispose for pathological processes, such as Alzheimer’s disease (AD) and depression. Lipids are a major constituent of the brain, and specifically signaling lipids have been shown to regulate brain function. Here, we used a mass spectrometry-based lipidomic approach to evaluate the impact of a chronic unpredictable stress paradigm on the rat brain in a region-specific manner. We found that the prefrontal cortex (PFC) was the area with the highest degree of changes induced by chronic stress. Although the hippocampus presented relevant lipidomic changes, the amygdala and to a more extent, the cerebellum, presented few lipid changes upon chronic stress exposure. The sphingolipid and phospholipid metabolism were profoundly affected, showing an increase in ceramide and a decrease in sphingomyelin and dihydrosphingomyelin levels, and decreased phosphatidylethanolamine and ether phosphatidylcholine and increased lysophosphatidylethanolamine levels, respectively. Furthermore, the fatty acyl profile of phospholipids and diacylglycerol revealed that chronic stressed rats had higher 38 carbon(38C)-lipid levels in the hippocampus and a decrease in 36C-lipid levels in the PFC. Finally, lysophosphatidylcholine levels in the PFC were found to be correlated with blood corticosterone levels. In summary, lipidomic profiling of the effect of chronic stress allowed for the identification of dysregulated lipid pathways, revealing putative targets for pharmacological intervention that may potentially be used to modulate stress-induced deficits.

Introduction

It is well established that prolonged exposure to stress triggers deleterious effects on brain structure and function. Indeed, animals exposed to chronic stress have functional and morphological impairment in various brain regions, such as the hippocampus, the prefrontal cortex (PFC) and the amygdala1, 2. The implications of these stress-induced alterations are multiple, with an impact on learning and memory,3, 4 decision making5 and emotional responses6. Moreover, stress has been linked to pathological processes involved in highly prevalent diseases, such as Alzheimer’s diseases (AD)7 and depression8. It is thus crucial to thoroughly understand the molecular basis underlying the pathogenic effects of chronic stress in order to identify novel targets for therapeutic interventions against stress-induced alterations.

Stress response is characterized by a fast activation of the sympathetic nervous system, followed by a slower activation of the hypothalamic-pituitary-adrenal axis that culminates with the release of glucocorticoids (GCs): cortisol in primates and corticosterone (CORT) in rodents. These hormones, believed to mediate a significant part of the pathologic effects of chronic stress, are derived from cholesterol and typically bind to intracellular receptors. There are two different corticosteroid receptors, the mineralocorticoid receptor (MR) and the GC receptor (GR), which, dimerize upon ligand binding and translocate to the nucleus, regulating gene transcription; the role of GCs receptors in the membrane has been recognized but their functional role is still being scrutinized9. Because CORT has a much higher affinity for MR than for GR, the latter is occupied only in the presence of elevated CORT levels, such as those triggered by chronic stress10. Importantly, it has been shown that CUS leads to major structural and functional hippocampal alterations4, 11, 12, which are, at least in part, mediated by increased levels of CORT9, 11. Therefore, it is critical to gain insights into the molecular mechanisms responsible for the brain manifestations of chronic stress and determine the contribution of CORT-dependent and -independent processes to these manifestations. Growing evidence suggests that stress/GCs may impact on brain lipid metabolism. For instance, GCs have been shown to modulate brain lipid signaling via stimulation of phospholipase A2 (PLA2), a key lipid enzyme that cleaves membrane phospholipids into lysolipids and arachidonic acid (AA), which are associated with inflammatory processes13. Importantly, it has been shown that chronic stress also alters the expression of a lipid modifying enzyme, diacylglycerol (DG) lipase α, thus modulating the levels of DG and 2-arachidonoylglycerol in stress-susceptible brain regions14. Moreover, chronic stress was shown to lead to an increase in ceramide (Cer) levels in the mouse hippocampus15.

Given the implication of multiple facets of lipid signaling in stress, we reasoned that a comprehensive mass spectrometry-based lipidomic analysis of rodent brain under conditions of stress would be both timely and informative. We evaluated the impact of a four week chronic unpredictable stress (CUS) paradigm on various rat brain regions believed to be affected by stress: hippocampus, PFC and amygdala, and, as a control, we analyzed the cerebellum, which is potentially a brain region not significantly affected by stress2. Overall, the PFC was the brain area with the highest degree of changes induced by chronic stress. Specifically, sphingolipid metabolism was affected, showing an increase in Cer and a decrease in sphingomyelin (SM) and dihydrosphingomyelin (dhSM) levels. Also, phospholipid metabolism was found to be altered, with decreased phosphatidylethanolamine (PE) and ether phosphatidylcholine (PCe) levels and an increase in lysophosphatidylethanolamine (LPE) levels. Furthermore, the fatty acid profile of phospholipids and DG revealed that chronically stressed rats had higher hippocampal 38 carbon (38C)-lipid levels and decreased PFC 36C-lipid levels. Finally, our lipidomic analysis revealed multiple brain lipid species levels to be correlated with blood CORT levels.

Methods

Animals and treatments

Experiments were conducted in accordance with local regulations (European Union Directive 86/609/EEC) and National Institutes of Health guidelines on animal care and experimentation. Adult (2 months old at the beginning of the experiment) male Wistar rats (Charles River Laboratories, Barcelona, Spain) were housed in groups of 2 under standard laboratory conditions (lights on from 8:00 A.M. to 8:00 P.M.; room temperature 22°C; ad libitum access to food and drink). In our experimental design we tested 4 different conditions. [1] A group of 10 rats were handled daily and served as controls. [2] A group of 10 rats were submitted to 4 weeks of a chronic unpredictable stress paradigm as described by Cerqueira et al3. Briefly, animals were exposed once daily to a stressor (1 h/d) of one of several aversive stimuli [cold water (18°C), vibration, restraint, overcrowding, exposure to a hot air stream]; the stressors were presented in random order for the duration of the experiment. [3] A group of 10 animals was used as a vehicle control group, with daily subcutaneous injections of sesame oil for 4 weeks. [4] A group of 10 animals underwent daily subcutaneous injections of synthetic CORT 40 mg/kg (Sigma-Aldrich) dissolved in sesame oil for 4 weeks. This CUS stress paradigm was previously shown to result in persistently elevated plasma levels of CORT, the primary GC of the rat.3

To ensure the effectiveness of the experimental procedures, serum CORT levels were measured using a commercially available radioimmunoassay kit (MP Biochemicals) after the 4 week treatment regimens (between 8:00 and 9:00 A.M.). The effectiveness of the CUS paradigm was confirmed with measurement of serum CORT levels, with a 58% increase in the CUS animals (control group 98,9 ng/ml ± 11,8; CUS group 154,1 ng/ml ± 11,8; p = 0,029), and with behavioral analysis using the elevated plus maze test (EPM) (see below for methodological details), with a 48.5% decrease in fraction of time spent in open arms for CUS animals (control group 38,1 sec ± 10,2; CUS group 19,6 ± 11,8; p = 0,022). One serum sample was lost during the experimental procedure.

Behavioral testing - Elevated Plus Maze

The elevated plus maze (EPM) behavioral paradigm was used to test anxiety-like behavior. The EPM apparatus was made of black polypropylene (ENV-560; MedAssociates), consisted of two opposite open arms (50.8 × 10.2 cm) and two enclosed arms (50.8 × 10.2 × 40.6 cm) elevated 72.4 cm above the floor. The junction area between the four arms measured 10 × 10 cm. A raised edge (0.5 cm) on the open arms provided additional grip for the rats. Rats were placed individually in the center of the maze facing a closed arm and were allowed 5 min of free exploration. Behavioral parameters were recorded with the use of an infra-red photobeam system connected to a computer with specific software (MedPCIV, MedAssociates) and posteriorly confirmed by video analysis. After each trial, the maze was cleaned with 10% ethanol. The percentage of time spent in the open arms was taken as an index of anxiety-like behavior.16

Annotation of Lipid Species

Lipids are annotated with subclass name followed by total fatty radyl carbons and unsaturation as needed. DG and TG species are annotated similarly, with the addition of fatty acyl carbon and unsaturation of the product ion. All measured sphingolipids contained d18:1 long-chain base (excepting dhSM species, containing a d18:0 base). Measured acyl-phosphatidylserine (APS) species all contained C16:0 N-linked fatty acyl chains.

Analysis of Lipids Using High Performance Liquid Chromatography-Mass Spectrometry

Upon completion of behavioral testing, the animals were killed, macrodissection dissection of the hippocampus, PFC, amygdala and cerebellum was performed and all tissue samples were immediately collected in tubes, drop-freezed in liquid nitrogen and subsequently stored at −80°C for further processing. Samples from each brain area were processed simultaneously. Tissue lipid extracts were prepared using a modified Bligh/Dyer procedure, spiked with appropriate internal standards, and analyzed using a 6490 Triple Quadrupole LC/MS system (Agilent Technologies, Santa Clara, CA). Glycerophospholipids and sphingolipids were separated with normal-phase HPLC as before17, 18, with a few changes. An Agilent Zorbax Rx-Sil column (inner diameter 2.1 × 100 mm) was used under the following conditions: mobile phase A (chloroform:methanol:1 M ammonium hydroxide, 89.9:10:0.1, v/v) and mobile phase B (chloroform:methanol:water:ammonium hydroxide, 55:39.9:5:0.1, v/v); 95% A for 2 min, linear gradient to 30% A over 18 min and held for 3 min, and linear gradient to 95% A over 2 min and held for 6 min. Sterols and glycerolipids were separated with reverse-phase HPLC using an isocratic mobile phase as before17 except with an Agilent Zorbax Eclipse XDB-C18 column (4.6 × 100 mm). Individual lipid species were measured by multiple reaction monitoring (MRM) transitions and lipid concentration was calculated by referencing to appropriate internal standards: D5-cholesterol, CE 17:0, 4ME 16:0 diether DG, D5-TG 16:0/18:0/16:0, PA 14:0/14:0, PC 14:0/14:0, PE 14:0/14:0, PG 15:0/15:0, PS 14:0/14:0, LPC 17:0, LPE 14:0, LPI 13:0, BMP 14:0/14:0, SM d18:1/12:0, dhSM d18:0/12:0, Cer d18:1/17:0, GalCer d18:1/12:0, LacCer d18:1/12:0, and Sulf d18:1/17:0 (Avanti Polar Lipids, Alabaster, AL). PI 16:0/16:0 was purchased separately (Echelon Biosciences, Salt Lake City, UT). Some lipid classes do not have commercially available internal standards so these lipids were referenced to standards that are closely eluted in the LC-MS method: Ether-linked species were normalized to corresponding acyl-linked standards, NAPS to PS 14:0/14:0, and GM3 to PI 16:0/16:0. Lipid concentration was normalized by molar concentration across all species for each sample, and final data are presented as mean mol %.

Gene expression measurements by qRT-PCR

Total RNA was extracted from pre-frontal cortex using the TripleXtractor (Grisp), following the manufacturer’s instructions. RNA quality and quantification was assessed in the NanoDrop ND-1000 (NanoDrop, Thermo Scientific, Wilmington, DE, USA) and a total of 1ug of RNA from each sample was reverse transcribed into cDNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer’s instructions. Primers used to measure the expression levels of selected mRNA transcripts of Rattus norvegicus and of Mus musculus (conserved sequence) by qRT-PCR were designed using the Primer3 software, on the basis of the respective GenBank sequences, confirming intron spanning using the BLAT software. The reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal standard for the normalization of the selected transcripts expression. All gene accession numbers and primer sequences are provided in Supplementary Table 1. qRT-PCR was performed on a CFX 96TM real-time system instrument (Bio-Rad Laboratories), with the SsoFastTM EvaGreen® Supermix (Bio-Rad Laboratories) according to the manufacturer’s instructions, using equal amounts of cDNA from each sample. The cycling parameters were 1 cycle at 95 C for 1 min, followed by 39 cycles at 95 C for 15 s, annealing temperature (primer specific) for 20 s and 72 C for 20 s, finishing with 1 cycle at 65 C to 95 C, with an increment of 0.5 C, for 5 s (melting curve). Product fluorescence was detected at the end of the elongation cycle. All melting curves exhibited a single sharp peak at the expected temperature.

Data Analysis and Presentation

All lipid species and subclasses analyzed were found to have equal variance (data not shown) then analyzed with an unpaired two-tailed Student’s t-test. CORT levels and EPM data were analyzed with an unpaired one-tailed Student’s t-test In all cases, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Correlation data analysis was performed using the linear regression model and coefficient of determination and values expressed as R2. Error bars represent the mean standard error (SEM) in all cases.

Results

Chronic Stress affects preferentially the Prefrontal Cortex lipidome

In the control group, different rat brain areas exhibited different lipidomic profiles in the absence of treatment. Considering the major lipid species, we observed that free cholesterol is the most abundant lipid in all the regions analyzed, presenting higher levels in the cerebellum and proportionally lower levels in the PFC. In contrast, PC, the second most abundant lipid, displayed an opposite pattern with relative lower levels in the cerebellum and higher levels in the PFC. On a second level of magnitude, SM, PE, plasmalogen phosphatidylethanolamine (PEp), PI, PS, Sulf, CE, DG, dhSM and HexCer presented moderately higher levels compared to the remainder minor subclasses evaluated (Figure 1).

Figure 1.

Figure 1

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Chronic Stress alters the lipid composition in the rat brain. Lipid composition of different areas of the brain of adult rats submitted to 4 weeks of chronic unpredictable stress (CUS, red bars) compared with control rats (black bars). Four brain regions were analyzed – hippocampus (a), prefrontal cortex (b), amygdala (c) and cerebellum (d). Values shown are normalized to measured molar lipid concentration. 24-OHC, 24(S)-hydroxycholesterol; CE, cholesteryl ester; DG, diacylglycerol; TG, triacylglycerol; Cer, ceramide; SM, sphingomyelin; dhSM, dihydrosphingomyelin; HexCer, hexosylceramide; Sulf, sulfatides; Sulf(2OH), 2-hydroxy N-acyl sulfatide; LacCer, lactosylceramide; GM3, monosialodihexosylganglioside; PA, phosphatidic acid; PC, phosphatidylcholine; PCe, ether phosphatidylcholine; PE, phosphatidylethanolamine; PEp, plasmalogen phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; LPC, lysophosphatidylcholine; LPCe, ether lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPEp, plasmalogen lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; BMP, bis(monoacylglycero)phosphate; APS, acyl-phosphatidylserine. N = 10 per group. Results are presented as mean and bars as standard error of the mean. * p ≤ 0,05; ** p ≤ 0,01; *** p ≤ 0,001 by unpaired two-tailed Student’s t-test.

Overall, the PFC was the area with the highest degree of changes induced by chronic stress, with sphingolipid metabolism being the most affected (Figure 1 and Supplementary Figure 1). Considering the total lipid species per lipid class, we found that, in the PFC, CUS led to a sphingolipid imbalance with an increase in Cer and lactosylceramide (LacCer) levels and a decrease in SM and dhSM levels. Other lipid classes were also affected, as observed by a decrease in PE and ether PC (PCe) levels and an increase in LPE and triacylglycerol (TG) levels (Figure 1 and Supplementary Figure 1). A more profound lipid species analysis of the major altered lipid subclasses (Cer, LacCer, SM, dhSM, and PE) in the PFC revealed that there was an overall coherent effect on the majority of the analyzed lipid species (Figure 2).

Figure 2.

Figure 2

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Chronic stress has a significant impact on sphingolipid and phosphatidylethanolamine metabolism in the prefrontal cortex. Prefrontal cortex lipid species composition of adult rats subjected to 4 weeks of chronic unpredictable stress (CUS, red bars) compared with control rats (black bars) submitted to handling. Analysis showed increases in several ceramide (a) and all lactosylceramide (b) species and decreases in many sphingomyelin (c), dihydrosphingomyelin (d), and phosphatidylethanolamine (e) species. y values expressed as relative levels to control animals. Cer, ceramide; LacCer, lactosylceramide; SM, sphingomyelin; dhSM, dihydrosphingomyelin; PE, phosphatidylethanolamine. n=10 per group. Results are presented as mean and bars as standard error of the mean. * p ≤ 0,05; ** p ≤ 0,01; *** p ≤ 0,001.

CUS led to a similarly altered sphingolipid pattern in the hippocampus, with an increase in Cer and a decrease in SM levels. Other lipid changes such as an increase in PI levels and decreased acyl-phosphatidylserine (APS) levels were also observed (Figure 1 and Supplementary Figure 1).

Interestingly, the amygdala presented only minor alterations, with increased levels of 2-hydroxy N-acyl sulfatide [Sulf(2OH)], a myelin lipid, and no significant differences were found in the cerebellum (Figure 1 and Supplementary Figure 1).

Fatty-acid lipid composition is affected by Chronic Stress

Brain function relies on the homeostasis of cellular membranes, and its perturbation might partially explain the neuronal deficits observed in various neurological conditions. Both the carbon chain length and the degree of saturation of the fatty acyls that are part of membrane lipids determine membrane biophysical properties. Therefore, the modulation of the composition of its fatty acyl chains can potentially affect neuronal functioning, at least in part through altered function of membrane-bound proteins.19, 20 In our study we performed another level of analysis focusing on the fatty acyl chain profile. Our data shows that CUS led to a significant alteration in the fatty acyl chain profile of phospholipids and DG, with increased levels of long chain fatty acyls with 38 carbons (38C) and an increase in polyunsaturated fatty acyls with 4 double bonds in the hippocampus (Figure 3). While in the PFC we observed a decrease in 36C fatty acyls, in the cerebellum we observed an increase in its levels. Interestingly, concerning the effects of CUS in sphingolipid composition, only the PFC was affected with an increase in short chain fatty acyls (16C) and a decrease in medium length (20C) fatty acyls (Figure 3).

Figure 3.

Figure 3

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Altered fatty acid composition after chronic unpredictable stress exposure (CUS) in different rat brain regions. Analysis of fatty acyl composition of diacyl-glycerophospholipids and DG species by total carbon chain length (a1, b1, c1, d1), total degree of unsaturation (a2, b2, c2, d2) and sphingolipid N-acyl chain lengths (a3, b3, c3, d3), Values are expressed as mol % + SEM of included lipid species. Hippocampus (a1-a3), prefrontal cortex (b1-b3), amygdala (c1-c3) and cerebellum (d1-d3) lipid tissue extracts were analyzed. Adult rats subjected to 4 weeks of chronic unpredictable stress (CUS, red bars) compared with control rats (black bars) submitted to handling. n = 10 per group. * p ≤ 0,05; ** p ≤ 0,01; *** p ≤ 0,001.

Specific lipid species are correlated with blood corticosterone levels after chronic stress exposure

The stress response is characterized by an acute activation of the sympathetic nervous system, followed by a slower activation of the hypothalamic-pituitary-adrenal axis that culminates with the release of GCs, such as cortisol in primates and corticosterone (CORT) in rodents. To test which lipid species were associated with serum CORT levels, we performed a full scale unbiased correlation analysis between serum levels of this hormone and abundance of the 359 quantifiable lipid species in each brain region. We defined a cut-off of correlation index of 0,4 (R2) in order to limit the hits in our analysis (Figure 4a). We found that CORT serum levels were directly correlated with PFC levels of total levels of lysophosphatidylcholine (LPC) (Figure 4b1) and inversely correlated with total SM levels (Figure 4b2). Moreover, various LPC species in the PFC, LPC 20:4 (Figure 4b3), LPC 20:3 (data not shown), LPC 16:0 (data not shown) and PA 40:5 (Figure 4b4) levels were found to be directly correlated with serum CORT levels.

Figure 4.

Figure 4

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Specific lipid species brain levels are directly correlated with blood corticosterone levels. (a) Correlation levels between all lipid species analyzed and blood corticosterone (CORT) levels. Lipid species are grouped per lipid class and rat brain areas are represented with different color dots, hippocampus (Hip - black), prefrontal cortex (PFC - red), amygdala (Amyg - grey) and cerebellum (Cereb - pink). R2 levels are represented from (−1) to (+1), with positive values indicating a positive correlation and negative values indicating a negative correlation. (b–e) For each brain area analyzed, prefrontal cortex (b), hippocampus (c) and amygdala (d), the major positive lipid species CORT-correlation hits are represented. Not all positive hits are represented in the figure. Adult rats submitted to 4 weeks of chronic unpredictable stress (CUS, red dots) and handled controls (black dots), animals are represented. (b1–4) Selected lipid species with a higher degree of correlation index in the prefrontal cortex. (c1) Selected lipid species with a higher degree of correlation index in the hippocampus. (d) Selected lipid species with a higher degree of correlation index in the amygdala. 24-OHC, 24(S)-hydroxycholesterol; CE, cholesteryl ester; DG, diacylglycerol; TG, triacylglycerol; Cer, ceramide; SM, sphingomyelin; dhSM, dihydrosphingomyelin; HexCer, hexosylceramide; Sulf, sulfatides; Sulf(2OH), 2-hydroxy N-acyl sulfatide; LacCer, lactosylceramide; GM3, monosialodihexosylganglioside; PA, phosphatidic acid; PC, phosphatidylcholine; PCe, ether phosphatidylcholine; PE, phosphatidylethanolamine; PEp, plasmalogen phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; LPC, lysophosphatidylcholine; LPCe, ether lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPEp, plasmalogen lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; BMP, bis(monoacylglycero)phosphate; APS, acyl-phosphatidylserine. n = 10 per group.

In the hippocampus we found a single correlation hit for LPC 20:4 (Figure 4c). In the amygdala LPC 20:4 (Figure 4e) was also found to be correlated with blood CORT levels, as well as phosphatidylserine (PS) 38:1 levels (data not shown). Finally, no correlation hits were found in the cerebellum.

Common lipid changes after chronic stress and exogenous corticosterone exposure

In order to gain further insight into which lipid species alterations induced by CUS are in fact mediated by CORT, we evaluated the impact of exogenous administration of CORT in the PFC lipid profile and the lipid induced alterations common to both the CUS group and CORT injected animals. We found a common increase in Cer 18:1, PG 36:1, phosphatidic acid (PA) 40:5, PA 40:6, LPC 16:0 and a common decrease in multiple PC, PCe and PE lipid species (Figure 5).

Figure 5.

Figure 5

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Common prefrontal cortex (PFC) lipid changes after chronic unpredictable stress (CUS) and exogenous corticosterone (CORT) exposure. Common altered lipid species composition in the prefrontal cortex of adult rats submitted to 4 weeks of CUS or (red bars) submitted to 4 weeks of subcutaneous CORT (40mg/kg) injections (pink bars). Results are expressed as percentual change ratio with each control group; for the CUS the ratio values are relative to control handled animals; for the CORT injected animals the ratio values are relative to vehicle injected animals. Cer, ceramide; PA, phosphatidic acid; PC, phosphatidylcholine; PCe, ether phosphatidylcholine; PE, phosphatidylethanolamine; PEp, plasmalogen phosphatidylethanolamine; PG, phosphatidylglycerol; LPC, lysophosphatidylcholine. N = 10 per group. Results are presented as mean and bars as standard error of the mean. * p ≤ 0,05; ** p ≤ 0,01; *** p ≤ 0,001.

Stress induced-lipid changes in the PFC are independent of changes in enzymatic expression levels

In light of our comprehensive lipid analysis, and according with previous studies showing in a mouse model of chronic stress had altered transcriptomic levels in the PFC21, one possibility was that the lipid changes we observed in the PFC after exposure to CUS, could be due to expression levels alterations of enzymatic lipid modulators. We measured mRNA levels of some of our most likely candidate hit list, namely, Smpd1 - acidic sphingomyelinase, Smpd2 - neutral sphingomyelinase, Pla2g2C, Pla2G4a, Pla2g5, Pla2g6, Pld1 and Pld2 (Supplementary Figure 2). We found no differences in the expression levels of our candidate enzymatic hits, suggesting that at least some of the lipid changes we found are independent of expression level regulation of the main catalytical enzymes of our candidate lipid pathways.

Discussion/Conclusions

In the present study we performed a detailed lipidomic analysis to understand the impact of chronic stress in distinct brain areas. So far, very few studies have analyzed the impact of either acute22 or chronic stress23, 24 in lipid signaling using a lipidomic approach, and predominantly the focus has been put on the role of endocannabinoids as modulators of stress associated pathways25.

This thorough analysis of the rodent brain lipidome unmasks the PFC as a key target of stress, with the highest degree of lipid changes induced by chronic stress. Specifically, in the PFC, sphingolipid metabolism was highly affected with increased Cer and decreased SM and dhSM levels, and phospholipid metabolism with decreased PE and increased LPE levels. This region vulnerability fits with previous studies showing that the PFC is the brain region displaying major lipid alterations after the use of maprotiline, an antidepressant26. In line with the PFC changes, we found that the hippocampus lipid profile also displayed sphingolipid metabolic changes and increased PI levels. The similar pattern of lipid changes is interesting, as previous studies demonstrated the PFC and the hippocampus to be functionally and structurally impaired after chronic stress exposure1, 2. The lipid alterations found in our study suggest that sphingolipid modulating enzymes may be dysregulated in both regions, either due to increased sphingomyelinase (SMase) activity (which catalyzes the hydrolysis of SM and dhSM into Cer and dhCer, respectively) or decreased SM synthase activity (which catalyzes the opposite reaction)27. Alternatively, other pathways for ceramide formation, such as de novo synthesis or hydrolysis of more complex sphingolipids, cannot be excluded as potential contributors to the observed increase in ceramide levels28. This is in accordance with what was shown in other studies where hippocampal levels of Cer were found to be increased in a CUS mouse model15. Importantly, these sphingolipid alterations can potentially alter the organization of specific lipid microdomains, such as lipid rafts, with implications for brain function29.

The combination of decreased PE and increased LPE levels suggests a hyperactive state of a PLA2 isoform30 in the PFC after CUS exposure. Interestingly, decreased levels and pharmacologic inhibition of acidic SMase prevented CUS induced alterations15, and the downregulation of calcium independent PLA2 (iPLA2) blocked both the behavior induced alterations and the PFC lipid profile due to maprotiline chronic treatment31. Also noticeable, is the observation that PLA2 activation is associated with increased inflammation. In a genetically induced model of neurodegeneration, it was shown that specific lysophospholipids were responsible for microglial activation and that cytosolic PLA2 (cPLA2) inhibition protected neurons from associated local inflammation and glutamate-mediated neurotoxicity32.

Besides comparing the individual species we also analyzed the fatty acyl chain profile of both phospholipids/DG and sphingolipids. In summary, we found that CUS led to a decrease in 36C fatty acyl phospholipids/DG levels in the PFC and to an increase in 38C and 4 double bond polyunsaturated phospholipids/DG levels in the hippocampus. Interestingly, the hippocampal data indicates the possible accumulation of phospholipids containing AA (20:4) as part of the fatty acyl composition. This is supported by findings in a depressive-like rat model where a similar pattern of increased levels of AA-containing phospholipids and decreased docosahexaenoic acid (DHA)-containing phospholipids occurs in the PFC33, 34. Also, diets rich in polyunsaturated fatty acids (PUFA), such as fish oil diet, showed an increase in DHA-containing phospholipids PC 40:6 and PC 38:6 and a decrease in AA-containing phospholipids PC 38:4 and PC 36:435. The DHA-treated rats had decreased levels of AA-containing phospholipids with concomitant prevention of the lipid alterations induced by aging36. Finally, diets deficient in n-3 PUFA led to decreased DHA levels and increased DHA half-life due to a downregulation of iPLA2 levels and upregulation of both secreted PLA2 (sPLA2) and cPLA2 in the frontal cortex37. Overall, our data, and the previous studies, suggest that AA accumulation in the hippocampus might be deleterious, while DHA accumulation in the frontal cortex might be protective in the context of neurodegeneration. Since it was also previously proposed that while cPLA2 preferentially releases AA and that iPLA2 preferentially cleaves DHA-containing phospholipids38, it is plausible that there is a dysregulation of iPLA2 in the PFC and of cPLA2 in the hippocampus (as it is discussed in37 and 38). Moreover, patients with depression were found to have increased ratio blood levels of n-6/n-3 PUFA39, and chronic stress in rodents altered lipoprotein blood and liver metabolism40, 41. Of notice, circulating FA can reach the brain and be incorporated in the cells of the central nervous system (CNS)42. Therefore, all these aspects are of critical relevance for our understanding of the lipid metabolism crosstalk between CNS and the periphery in physiological and pathological conditions, and should be addressed in future studies.

Since elevations in CORT levels are a critical element in the stress response, we tested which of the stress effects in brain lipidomics could be attributed to hypercortisolemia. Firstly, with an unbiased strategy, we identified which lipid species had a higher degree of correlation with endogenous CORT levels; secondly, we identified the common alterations in the PFC lipid profile of both CUS and CORT injected animals. In the PFC we found that CORT serum levels were directly correlated in the PFC with multiple LPC species levels, and inversely correlated with total SM levels. Interestingly, we found that, specifically, LPC 20:4 was found as a positive correlation hit in all brain areas studied except the cerebellum. This is in accordance with the also observed accumulation of AA containing phospholipids, which is thought to have a deleterious effect. Among the few studies addressing the role of LPC 20:4, it was shown that it can promote inflammatory events through the donation of AA, which is proposed to be released in a cPLA2 dependent way43. Moreover, both CUS and CORT injected animals had increased levels of Cer 18:1 and LPC 16:0 and decreased levels in multiple PC, PCe and PE species, suggesting that CORT might be leading to an activation of PLA2 and SMase in the PFC of stressed animals. Interestingly, PA 40:5 PFC levels were directly correlated with endogenous blood CORT levels, and PA 40:5 and PA 40:6 levels were both increased in the PFC of CUS and CORT injected animals, which suggests that phospholipase D (PLD)-dependent-pathways might also be activated by GCs. This is in accordance with previous reports showing increased PLD activity after treatment with a synthetic corticosteroid44, the latter is likely to result from the GC induced calcium influx45, a known activator of PLD46. Interestingly, PLD activation was also shown to be detrimental in other neurodegeneration conditions47, 48.

In summary, herein we identified the lipid alterations induced by chronic stress and CORT in target brain areas. Our data shows that the metabolism of sphingolipids and phospholipids is significantly altered by stress in both the PFC and the hippocampus, suggesting SMase, PLA2 and PLD as potential therapeutical targets for stress-related disorders. These alterations in lipid signaling pathways have several implications in physiological mechanisms, such as, neurogenesis and synaptic plasticity, known to be implicated in several stress-related disorders, such as emotional disorders15, 49–51, and eventually other psychiatric disorders, such as schizophrenia51–54. Given that there are different SMase, PLA2 and PLD isozymes, future lipidomic studies addressing the impact of lipid signaling-targeted therapeutical drugs will provide the pharmacological mechanistic insights in order to choose the best candidate drug in a given pathological condition. Also, other non-pharmacological approaches, such as lifestyle changes and therapeutic diets with previously predetermined brain lipid profiles may prove of interest in these conditions.

Supplementary Material

Suppl Figures

Supplementary Figure 1 Chronic Stress (CUS) preferentially affects the rat prefrontal cortex lipid profile. Ratio of lipid levels of rats submitted to 4 weeks of chronic unpredictable stress compared with control handled animals. The data is expressed as log2-normalized values relative to each control group (red - higher values; blue - lower values). Only significant changes, i.e., p < 0,05, are shown. 24-OHC, 24(S)-hydroxycholesterol; CE, cholesteryl ester; DG, diacylglycerol; TG, triacylglycerol; Cer, ceramide; SM, sphingomyelin; dhSM, dihydrosphingomyelin; HexCer, hexosylceramide; Sulf, sulfatides; Sulf(2OH), 2-hydroxy N-acyl sulfatide; LacCer, lactosylceramide; GM3, monosialodihexosylganglioside; PA, phosphatidic acid; PC, phosphatidylcholine; PCe, ether phosphatidylcholine; PE, phosphatidylethanolamine; PEp, plasmalogen phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; LPC, lysophosphatidylcholine; LPCe, ether lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPEp, plasmalogen lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; BMP, bis(monoacylglycero)phosphate; APS, acyl-phosphatidylserine. n = 10 per group.

Supplementary Figure 2 Prefrontal cortex chronic stress induced-lipid changes are independent of enzymatic expression levels modulation. mRNA levels of Smpd1 - acidic sphingomyelinase; Smpd2 - neutral sphingomyelinase; Pla2g2c – phospholipase A2 group IIC; Pla2GIVA - phospholipase A2 group IVA; Pla2GV - phospholipase A2 group V; Pla2GVI - phospholipase A2 group VI; Pld1 – phospholipase D1; and Pld2 – phospholipase D2, using prefrontal (PFC) extracts from rats after exposure to 4-week chronic unpredictable stress (CUS). N = 10 per group. Results are presented as mean and bars as standard error of the mean.

Acknowledgments

Funding by Fundação para a Ciência e Tecnologia (PTDC/SAU-NMC/118971/2010) and by the North Region Operational Program (ON.2 - O Novo Norte), under Quadro de Referência Estratégico Nacional (QREN) and through Fundo Europeu de Desenvolvimento Regional (FEDER). G.D.P is funded by NIH grants R01 NS056049 and P50 AG008702 (to Scott Small).

Footnotes

Conflict of interest

The authors declare no conflict of interest.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suppl Figures

Supplementary Figure 1 Chronic Stress (CUS) preferentially affects the rat prefrontal cortex lipid profile. Ratio of lipid levels of rats submitted to 4 weeks of chronic unpredictable stress compared with control handled animals. The data is expressed as log2-normalized values relative to each control group (red - higher values; blue - lower values). Only significant changes, i.e., p < 0,05, are shown. 24-OHC, 24(S)-hydroxycholesterol; CE, cholesteryl ester; DG, diacylglycerol; TG, triacylglycerol; Cer, ceramide; SM, sphingomyelin; dhSM, dihydrosphingomyelin; HexCer, hexosylceramide; Sulf, sulfatides; Sulf(2OH), 2-hydroxy N-acyl sulfatide; LacCer, lactosylceramide; GM3, monosialodihexosylganglioside; PA, phosphatidic acid; PC, phosphatidylcholine; PCe, ether phosphatidylcholine; PE, phosphatidylethanolamine; PEp, plasmalogen phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; LPC, lysophosphatidylcholine; LPCe, ether lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPEp, plasmalogen lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; BMP, bis(monoacylglycero)phosphate; APS, acyl-phosphatidylserine. n = 10 per group.

Supplementary Figure 2 Prefrontal cortex chronic stress induced-lipid changes are independent of enzymatic expression levels modulation. mRNA levels of Smpd1 - acidic sphingomyelinase; Smpd2 - neutral sphingomyelinase; Pla2g2c – phospholipase A2 group IIC; Pla2GIVA - phospholipase A2 group IVA; Pla2GV - phospholipase A2 group V; Pla2GVI - phospholipase A2 group VI; Pld1 – phospholipase D1; and Pld2 – phospholipase D2, using prefrontal (PFC) extracts from rats after exposure to 4-week chronic unpredictable stress (CUS). N = 10 per group. Results are presented as mean and bars as standard error of the mean.

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