Et group. -Soluble NSF protein showed slight downregulation in the 3 week samples of perilla-diet group but up-regulation in the 3 month samples of both perillaand perilla oil-diet groups. Overall, the fold changes of expression level in AMPA receptor, -synuclein, neurofilament, and -soluble NSF protein were correlated with the proteomic data and were always greater in the perilla oil-diet group compared to perilla-diet group in Western blotting analysis.Improved spatial learning and memory are associated with increased immunoreactivity to -synuclein and neurofilament in dentate gyrusAs AMPA receptor, neurofilament and -synuclein were all significantly up-regulated in the hippocampus of ratsperilla-diet intake, we questioned whether or not the upregulation of protein expression had any histological effects on the hippocampus. To test this, we used primary antibody against AMPA receptor as a positive control for improved learning and memory function along with antibodies against -synuclein, and neurofilament. We also performed immunohistochemistry staining on the sagittal microtome sections of rat brains (Figure 4). Tissue sections were prepared after completion of the behavioral studies. Anti-AMPA receptor antibody decorated the dentate gyrus of rats with significantly higher intensities following perilla-diet intake for 3 weeks or 3 months (Figure 5A) in comparison with control Vandetanib rats, suggesting that the rats indeed experienced improved spatial cognitive function by perilla-diets. Likewise, antibody against -synuclein showed higher immunoreactivity in the dentate gyrus of ratsfed perilla- or perilla- oil-diet for 3 weeks or 3 months (Figure 5B). However, no significant differences were observed in theLee et al. Proteome Science 2012, 10:72 http://www.proteomesci.com/content/10/1/Page 6 ofTable 3 Functional annotation clustering of the proteins differentially expressed in the hippocampus of rats fed perilla-dietsAnnotation cluster 3 week-feeding cytoskeleton membrane-bounded vesicle vesicle-mediated transport 3 month-feeding vesicle-mediated transport cytoskeleton neuron projection regulation of apoptosis 20 major proteins neurofilament cytoskeleton organization glycolysis regulation of axonogenesis 3 3 3 3.32 1.83 1.6 6.70E-05 1.80E-03 4.00E-03 8 9 7 7 2.28 2.24 2.17 2.1 1.20E-05 4.90E-04 2.40E-04 1.30E-03 9 7 7 2.97 1.79 1.4 9.20E-04 9.90E-04 1.70E-04 Number of proteins Enrichment score p-valueCA1 to 3 regions in the hippocampus or in the cortex (data not shown). Contrary to the results of the proteomic (Table 1) and the Western blotting analyses (Figure 3) of the hippocampus, no significant differences in neurofilament M were detected between the two groups in terms of immunostaining intensitied (quantified as intensity of immunostained cells/mm2 dentate gyrus: control=12.0961; perilla=12.2860; perilla oil=12.2899, Figure 4C) in the dentate gyrus. However, cells stained by the antineurofilament M antibody showed different cell morphologies. Immunoreactive cells in the hilus of dentate gyrus of the control group showed less neurite projections compared to cells of the perilla-diet groups (Figure 4). To observe the projections, high magnification photomicrographs PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 (representative images shown in (Figure 4C)) were taken, and the projections are indicated with arrows. The relative length of the projections measured as described in the Methods (Figure 5C) significantly increased in the perilla-diet groups, suggesting enhanced neuron project.