The impact of chronic stress on the rat brain lipidome

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 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 (CUS) 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 greater extent, the cerebellum presented few lipid changes upon chronic stress exposure. The sphingolipid and phospholipid metabolism were profoundly affected, showing an increase in ceramide (Cer) and a decrease in sphingomyelin (SM) and dihydrosphingomyelin (dhSM) levels, and a decrease in phosphatidylethanolamine (PE) and ether phosphatidylcholine (PCe) and increase in lysophosphatidylethanolamine (LPE) 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 reduced 36C-lipid levels in the PFC. Finally, lysophosphatidylcholine (LPC) levels in the PFC were found to be correlated with blood corticosterone (CORT) levels. In summary, lipidomic profiling of the effect of chronic stress allowed the identification of dysregulated lipid pathways, revealing putative targets for pharmacological intervention that may potentially be used to modulate stress-induced deficits.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.