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, 128 (4), 491-506

Brain Region-Specific Changes in N-acylethanolamine Contents With Time of Day

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Brain Region-Specific Changes in N-acylethanolamine Contents With Time of Day

Elizabeth Sabens Liedhegner et al. J Neurochem.

Abstract

The N-acylethanolamines (NAEs) exert important behavioral, physiological, and immunological effects through actions at cannabinoid and other receptors. We measured concentrations of three NAEs, the Km and Vmax for fatty acid amide hydrolysis (FAAH), FAAH protein and FAAH mRNA in prefrontal cortex, hippocampus, hypothalamus, amygdala, striatum, and cerebellum at 4 h intervals, starting at 03:00. Significant differences in N-arachidonylethanolamine contents among the times examined occur in the prefrontal cortex (PFC), hippocampus, hypothalamus, and striatum. N-Oleoylethanolamine concentrations exhibit large fluctuations over the day in the cerebellum, including a threefold decrease between 19:00 and 23:00. N-Palmitoylethanolamine and N-oleoylethanolamine were significantly, positively correlated in all regions examined except the hypothalamus. FAAH Km values are significantly affected by time of day in PFC, hippocampus and amygdala and FAAH Vmax values are significantly affected in PFC, hippocampus and cerebellum. However, correlational data indicate that FAAH does not play a primary role in the circadian regulation of the NAE concentrations. FAAH protein expression is not significantly different among the harvest times in any brain region examined. Concentrations of 2-arachidonoylglycerol are significantly affected by time of harvest in the striatum and cerebellum, but not in other brain regions. Together, these data indicate that the NAEs exhibit diverse patterns of change with time of day that are likely the result of alterations in biosynthesis, and support the hypothesis that N-arachidonylethanolamine is a tonic activator of cannabinoid receptor signaling.

Keywords: 2-arachidonoylglycerol; N-arachidonylethanolamine; N-oleoylethanolamine; circadian; fatty acid amide hydrolase; palmitoylethanolamide.

Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1
Effects of time of day on PFC contents of AEA (A), PEA (B) OEA (C) and 2-AG (D) were determined using isotope dilution, LC/MS. Each point is the mean of 6–9 determinations; vertical lines represent SEM. Two cycles of the same data are displayed. For AEA, PEA and OEA, Bonferroni’s t-tests were used to determine differences among harvest times; significant differences are noted; * p<0.05; ** p<0.01 and *** p<0.005. Correlations between AEA and PEA (E); AEA and OEA (F) and PEA and OEA (G) were examined using Pearson’s correlational analyses. The results of these analyses are reported in the text.
Figure 2
Figure 2
Effects of time of day on FAAH activity and expression in the PFC were determined. FAAH activity was measured as the conversion of AEA to ethanolamine in membranes harvested at the times indicated; Km and Vmax values were determined from isotherms created from 8 concentrations of AEA. Mean values of Km (A) and Vmax (B) are shown (n=4–5); vertical lines indicate SEM. Bonferroni’s t-tests were used to compare all values with each other; significantly different values are indicated; * p<0.05. (C) Western blot analyses were used to examine FAAH expression following separation of membrane proteins using gel electrophoresis. FAAH density was normalized to the density of GAPDH determined in the same sample. Each point is the mean of 4–6 separate tissues; vertical lines represent SEM. (D) mRNA for FAAH was determined in PFC harvested at the times shown using qPCR. Relative expression was calculated using GAPDH as a calibrator. Each point is the mean of 5–6 replicates, vertical lines represent SEM. For each graph, two cycles of the same data are displayed.
Figure 3
Figure 3
Effects of time of day on hippocampal contents of AEA (A), PEA (B) OEA (C) and 2-AG (D) were determined using isotope dilution, LC/MS. Each point is the mean of 5–9 determinations; vertical lines represent SEM. Two cycles of the same data are displayed. For AEA, PEA and OEA, Bonferroni’s t-tests were used to determine differences among harvest times; significant differences are noted; * p<0.05; ** p<0.01. Correlations between AEA and PEA (E); AEA and OEA (F); PEA and OEA (G) and PEA and 2-AG (H) were examined using Pearson’s correlational analyses. The results of these analyses are reported in the text.
Figure 4
Figure 4
Effects of time of day on FAAH activity and expression in the hippocampus were determined. FAAH activity was measured as the conversion of AEA to ethanolamine in membranes harvested at the times indicated; Km and Vmax values were determined from isotherms created from 8 concentrations of AEA. Mean values of Km (A) and Vmax (B) are shown (n=4–5); vertical lines indicate SEM. Bonferroni’s t-tests were used to compare all values with each other; significantly different values are indicated; * p<0.05; ** p<0.01 and *** p<0.005. In panel B, * p<0.05; ** p<0.01 and *** p<0.005 refer to differences from Vmax in tissue harvested at 0700; † p<0.05; †† p<0.01 and ††† p<0.005 compared to Vmax in tissue harvested at 0300. (C) Western blot analyses were used to examine FAAH expression following separation of membrane proteins using gel electrophoresis. FAAH density was normalized to the density of GAPDH determined in the same sample. Each point is the mean of 4–6 separate tissues; vertical lines represent SEM. (D) mRNA for FAAH was determined in PFC harvested at the times shown using qPCR. Relative expression was calculated using GAPDH as a calibrator. Each point is the mean of 5–6 replicates, vertical lines represent SEM. For each graph, two cycles of the same data are displayed. ** p<0.01 by Bonferroni’s t-test.
Figure 5
Figure 5
Effects of time of day on hypothalamic contents of AEA (A), PEA (B) OEA (C) and 2-AG (D) were determined using isotope dilution, LC/MS. Each point is the mean of 5–9 determinations; vertical lines represent SEM. Two cycles of the same data are displayed. For AEA, Bonferroni’s t-tests were used to determine differences among harvest times; significant differences are noted; ** p<0.01. Correlations between AEA and PEA (E); AEA and OEA (F) and PEA and OEA (G) were examined using Pearson’s correlational analyses. The results of these analyses are reported in the text.
Figure 6
Figure 6
Effects of time of day on FAAH activity and expression in the hypothalamus were determined. FAAH activity was measured as the conversion of AEA to ethanolamine in membranes harvested at the times indicated; Km and Vmax values were determined from isotherms created from 8 concentrations of AEA. Mean values of Km (A) and Vmax (B) are shown (n=4–5); vertical lines indicate SEM. (C) Western blot analyses were used to examine FAAH expression following separation of membrane proteins using gel electrophoresis. FAAH density was normalized to the density of GAPDH determined in the same sample. Each point is the mean of 4–6 separate tissues; vertical lines represent SEM. (D) mRNA for FAAH was determined in PFC harvested at the times shown using qPCR. Relative expression was calculated using GAPDH as a calibrator. Each point is the mean of 5–6 replicates, vertical lines represent SEM. For each graph, two cycles of the same data are displayed. * p<0.05 by Bonferroni’s t-test.
Figure 7
Figure 7
Effects of time of day on striatal contents of AEA (A), PEA (B) OEA (C) and 2-AG (D) were determined using isotope dilution, LC/MS. Each point is the mean of 5–9 determinations; vertical lines represent SEM. Two cycles of the same data are displayed. For AEA, PEA and 2-AG, Bonferroni’s t-tests were used to determine differences among harvest times; significant differences are noted; * p<0.05; ** p<0.01; ***p<0.005. Correlations between AEA and PEA (E); AEA and OEA (F) and PEA and OEA (G) were examined using Pearson’s correlational analyses. The results of these analyses are reported in the text.
Figure 8
Figure 8
Effects of time of day on FAAH activity and expression in the striatum were determined. FAAH activity was measured as the conversion of AEA to ethanolamine in membranes harvested at the times indicated; Km and Vmax values were determined from isotherms created from 8 concentrations of AEA. Mean values of Km (A) and Vmax (B) are shown (n=4–5); vertical lines indicate SEM. (C) Western blot analyses were used to examine FAAH expression following separation of membrane proteins using gel electrophoresis. FAAH density was normalized to the density of GAPDH determined in the same sample. Each point is the mean of 4–6 separate tissues; vertical lines represent SEM. (D) mRNA for FAAH was determined in PFC harvested at the times shown using qPCR. Relative expression was calculated using GAPDH as a calibrator. Each point is the mean of 5–6 replicates, vertical lines represent SEM. For each graph, two cycles of the same data are displayed. * p<0.05 by Bonferroni’s t-test.
Figure 9
Figure 9
Effects of time of day on amygdalar contents of AEA (A), PEA (B) OEA (C) and 2-AG (D) were determined using isotope dilution, LC/MS. Each point is the mean of 5–9 determinations; vertical lines represent SEM. Two cycles of the same data are displayed. For PEA and OEA, Bonferroni’s t-tests were used to determine differences among harvest times; significant differences are noted; * p<0.05; ** p<0.01. Correlations between AEA and PEA (E); AEA and OEA (F) and PEA and OEA (G) were examined using Pearson’s correlational analyses. The results of these analyses are reported in the text.
Figure 10
Figure 10
Effects of time of day on FAAH activity and expression in the amygdala were determined. FAAH activity was measured as the conversion of AEA to ethanolamine in membranes harvested at the times indicated; Km and Vmax values were determined from isotherms created from 8 concentrations of AEA. Mean values of Km (A) and Vmax (B) are shown (n=4–5); vertical lines indicate SEM. Bonferroni’s t-tests were used to compare all values with each other; significantly different values are indicated; * p<0.05. (C) Western blot analyses were used to examine FAAH expression following separation of membrane proteins using gel electrophoresis. FAAH density was normalized to the density of GAPDH determined in the same sample. Each point is the mean of 4–6 separate tissues; vertical lines represent SEM. (D) mRNA for FAAH was determined in PFC harvested at the times shown using qPCR. Relative expression was calculated using GAPDH as a calibrator. Each point is the mean of 5–6 replicates, vertical lines represent SEM. For each graph, two cycles of the same data are displayed.
Figure 11
Figure 11
Effects of time of day on cerebellar contents of AEA (A), PEA (B) OEA (C) and 2-AG (D) were determined using isotope dilution, LC/MS. Each point is the mean of 5–9 determinations; vertical lines represent SEM. Two cycles of the same data are displayed. For OEA, Bonferroni’s t-tests were used to determine differences among harvest times; significant differences are noted; * p<0.05; ** p<0.01; **** p<0.0001. Correlations between AEA and PEA (E); AEA and OEA (F) and PEA and OEA (G) were examined using Pearson’s correlational analyses. The results of these analyses are reported in the text.
Figure 12
Figure 12
Effects of time of day on FAAH activity and expression in the cerebellum were determined. FAAH activity was measured as the conversion of AEA to ethanolamine in membranes harvested at the times indicated; Km and Vmax values were determined from isotherms created from 8 concentrations of AEA. Mean values of Km (A) and Vmax (B) are shown (n=4–5); vertical lines indicate SEM. (C) Western blot analyses were used to examine FAAH expression following separation of membrane proteins using gel electrophoresis. FAAH density was normalized to the density of GAPDH determined in the same sample. Each point is the mean of 4–6 separate tissues; vertical lines represent SEM. (D) mRNA for FAAH was determined in PFC harvested at the times shown using qPCR. Relative expression was calculated using GAPDH as a calibrator. Each point is the mean of 5–6 replicates, vertical lines represent SEM. For each graph, two cycles of the same data are displayed.

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