A synaptogenic amide N-docosahexaenoylethanolamide promotes hippocampal development

Abstract

Docosahexaenoic acid (DHA), the n-3 essential fatty acid that is highly enriched in the brain, increases neurite growth and synaptogenesis in cultured mouse fetal hippocampal neurons. These cellular effects may underlie the DHA-induced enhancement of hippocampus-dependent learning and memory functions. We found that N-docsahexaenoylethanolamide (DEA), an ethanolamide derivative of DHA, is a potent mediator for these actions. This is supported by the observation that DHA is converted to DEA by fetal mouse hippocampal neuron cultures and a hippocampal homogenate, and DEA is present endogenously in the mouse hippocampus. Furthermore, DEA stimulates neurite growth and synaptogenesis at substantially lower concentrations than DHA, and it enhances glutamatergic synaptic activities with concomitant increases in synapsin and glutamate receptor subunit expression in the hippocampal neurons. These findings suggest that DEA, an ethanolamide derivative of DHA, is a synaptogenic factor, and therefore we suggest utilizing the term 'synaptamide'. This brief review summarizes the neuronal production and actions of synaptamide and describes other N-docosahexaenoyl amides that are present in the brain.

Fig. 1

Effects of inhibitors on DHA-induced development of mouse hippocampal neurons. Hippocampal cultures were prepared from fetuses obtained on embryonic day 18 (E18) from timed pregnant C57/BL6 mice and maintained in Neurobasal medium with 2 % B27 supplement as previously described [3,22]. The cultures then were treated with DHA (1 μM) for 7 days in the presence or absence of the fatty acid amide hydrolase inhibitor URB597 (FAAHI, 1 μM), cyclooxygenase inhibitor indomethacin (50 μM) or lipoxygenase inhibitor NDGA (10 μM). Representative photomicrographs are shown at 60× magnification. Corresponding control cultures were included. After incubation, neurons were stained with MAP2 (green, a neuron-marker protein), DAPI (blue, nuclear-marker) and synapsin 1 (red). These photomicrographs are taken from reference 22.

Fig. 2

Conversion of DHA to DEA in hippocampal cultures. A. The tandem mass (MS/MS) spectrum was obtained from [M+H]⁺ of ¹³C₂₂-DEA produced by mouse E-18 hippocampal neurons cultured for 3 days in the presence of 1 μM ¹³C₂₂-DHA. The characteristic fragmentation pattern indicates that ¹³C₂₂-DEA is produced by the cells. B. The MRM chromatograms using characteristic mss transitions from [M+H]⁺ to the ethanolamine moiety at m/z 62 confirmed the production of DEA in E-18 hippocampal cultures after supplementation with 1 μM ¹³C₂₂-DHA or for 3 days. The retention time of ¹³C₂₂-DEA is identical to that of the standard DEA. The mass spectrum is taken from reference 22

Fig. 3

Comparative effects of DHA and synaptamide on hippocampal neurite growth. A. Mouse E-18 hippocampal neurons were incubated for 3 days with 0.05 μM synaptamide, 0.5 μM DHA, or 0.1 μM 9-cis-retinoic acid (9C-RA), and immunostained for MAP2. Representative photomicrographs from at least 8 fields in each case are shown at 20× magnification. B. Total neurite length per neuron was measured in the neurons cultured for 3 days with DHA or synaptamide. The concentration range for DHA and synaptamide was 0.1 - 1 μM and 0.01 - 1 μM, respectively. Measurements of 60 neurons in each culture were made, and the statistical analysis was performed with the student’s t-test. The data presented are the mean values ± standard deviation, and the significant differences were compared to the control cultures: *, p < 0.05, and **, p < 0.05.

Fig. 4

Effects of synaptamide (0.1 μM) on neurite growth, synaptogenesis and synaptic activity. Representative photomicrographs are shown at 60× magnification. Synaptogenesis was evaluated by the number of synapsin puncta / 10μm neurite. **, p<0.01; ***, p<0.001. For synaptic activity, spontaneous postsynaptic currents (sPSCs) including glutamatergic (Glu-sPSCs) and GABAergic (GABA-sPSCs) components were recorded in hippocampal neurons cultured with or without 0.1 μM synaptamide for 10 days. To isolate the GABAergic sPSCs, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX, 5 μM) and D-2-amino-5-phosphonopentanoic acid (AP5, 50 μM) were used. For measurement of glutamatergic sPSCs, bicuculline (20μM) was superfused onto the neuron. Paired t-tests were performed against the baseline value of each group (##, p<0.01; ###, p<0.001) or between indicated groups (***, p<0.001). The bar graphs are taken from reference 22.

Fig. 5

Effects of N-acylethanolamides on neurite growth and synaptogenesis. Embryonic hippocampal neurons were cultured for 3 (A) or 7 days (B) with 0.1 μM synaptamide, AEA or OEA at concentrations from 1 to 5 μM. Representative photomicrographs are shown for neurons triple-stained with MAP2 (green), synapsin (red) and nuclei (blue). Scale bar: 30μm. While 0.1 μM synaptamide significantly increased neurite growth and synaptogenesis, AEA or OEA showed no effects at substantially higher concentrations.