As synapses are labile and plastic structures, any micro-environmental changes in the brain can alter the number and structural configurations of existing synapses. Changes in neurological signaling by alterations of the gene expression profiles and lipid composition in the synaptic membrane can modulate cognitive function in the brain
[18, 19]. Thus, the goals of this study were to analyze proteomic changes in the brain upon supplementation of α-linolenic acid, an n-3 fatty acid, rich perilla, or perilla oil as well as to identify proteins closely related to the cognitive function.
Short term (for 3 weeks) feeding of 18:3n-3 fatty acid-rich perilla-diets to rats significantly improved spatial learning memory (Figure
1), changed the level of 22:6n-3 fatty acid, DHA, in the brain (Figure
2 and Additional file
1), and altered proteome profiles compared to the corn-oil based control diet group (Tables
2). These changes were also observed in the rat groups fed perilla-diets long term (for 3 months). From the proteomic analysis, we found that the differentially expressed proteins in the hippocampus of rats fed perilla-diets for 3 weeks were mainly annotated into several functional groups, including cytoskeleton and transport, whereas the major functional groups of those proteins from the 3 month groups were energy metabolism, neuron projection and apoptosis in addition to transport and cytoskeleton (Table
3). The annotation grouping in this study shows that the changes in fatty acid composition in the membrane of the brain of rats fed n-3 fatty acid rich perilla-diets altered the expression profiles of hippocampal proteins, including cytoskeleton and energy metabolism proteins, hereby affecting neurogenesis and synaptic plasticity. Further, changes in the protein expression profile grew more complex as the animals became older, suggesting that there are common and specific pathways for cognition in response to various stimuli, and the pathways involved in cognitive function were much more complicated for the mature brain than for the young one. Previously, we demonstrated that estrogen depravation in rats by ovariectomy results in alleviated spatial learning and memory in a T-maze test, and we observed significant proteomic changes in energy metabolism, cytoskeleton, and anti-oxidation
. These results indicate that cytoskeleton as a common ground in conjunction with other proteins from different functional groups, improves cognition through various changes in synaptic plasticity.
To understand how the differentially expressed proteins are involved in synaptic plasticity, and based on the fold differences in 2-D gel electrophoresis, we selected four proteins for verification and functional analysis of the proteomic data. Antibodies against AMPA receptor, neurofilament, a-synuclein, and β-soluble NSF attachment protein were used for Western blotting and immunohistochemistry analysis.
Firstly, Western blotting analysis using antibodies against the selected proteins were performed (Figure
3). Although AMPA receptor could not be detected by 2-D gel electrophoresis and its differential expression in the hippocampus was not clear, it was selected as a positive control since the receptor subunits are essential for long term potentiation induction and maintenance
 and play important roles in spatial learning and memory
. Using anti-AMPA antibody, rats fed perilla-diets for 3 weeks or 3 months showed elevated AMPA receptor expression in the hippocampus, indicating that, together with the results from the T-maze test, n-3 fatty acid rich perilla-diets improved cognitive functions. Among the selected differentially expressed proteins in Tables
2, α-synuclein and neurofilament showed significantly increased expression, whereas β-soluble NFS attachment protein underwent slight changes in expression between the control group and perilla-diet groups in Western blotting analysis (Figure
3B). Further, the up-regulated proteins in the 2-D gel analysis showed higher expression in the Western blotting analysis. Interestingly, the perilla-diet group always showed lower protein expression than the perilla oil-diet group. This could be due to the higher availability of n-3 fatty acids in the digestive tract in the perilla oil group compared to perilla group (Figure
2). Oils in perilla seeds should be less available to the digestive enzyme system and thus less miscible and less absorptive in the intestine compared to the extracted perilla oil.
Secondly, using brain sections and antibodies whose target proteins were the most differentially expressed in the Western blotting analysis, we performed immunohistochemistry to examine where the proteins were expressed in the hippocampus as well as any changes in cell morphology. In addition, the specificity and reactivity of the available antibodies to the paraffin sections were considered.
α-Synuclein gene, which encodes a 140-amino-acid protein, is a key player in neurodegenerative diseases. The fibrillar β-sheet aggregation of α-synuclein is the major constituents of Lewy bodies and Lewy neurites in the pathogenesis of Parkinson disease
. However, in comparison to its identified roles in neuropathogenesis, the normal physiological function of α-synuclein in neurogenesis is not well understood. As a presynaptic protein, α-synuclein, is located in the membrane, interacts with tubulins, is enriched in presynaptic termini, and is involved in microtubule transport and neurotransmitter release
[22–24]. Mice lacking α-synuclein gene show normal long-term potentiation (LPT) of glutamatergic synapses in the hippocampus, suggesting that α-synuclein is not essential for LTP in the hippocampus
. Nevertherless, the modulation of α-synuclein gene expression in transgenic mice alters hippocampal neurogenesis and synaptic plasticity in the dentate gyrus, suggesting that the protein might have a relationship with learning and memory
[25, 26]. Interestingly, it was reported that an increasing concentration of DHA in the membrane of the brain induces aggregation and chemical modification of α-synuclein in vitro
. Regarding the fact that the perilla-diet groups showed elevated DHA levels in the brain (Figure
2 and Additional file
1), n-3 fatty acid converted to DHA could modulate the redistribution of α-synuclein in the hippocampal presynapse for synaptic plasticity and improved cognitive function. This result supports the previous finding that the mRNA level of α-synuclein was elevated upon n-3 fatty acid treatment to rats for 3 weeks
Neurofilament medium molecular weight protein (NF-M) is a neuron-specific intermediate filament protein that heteropolymerizes with other immunologically distinct neurofilaments, NF-L and NF-H, and constitutes a major part of the neuronal cytoskeleton
. The three different NF proteins form filaments at different stoichiometries under different physiological conditions, and the resulting sidearm structures of the C-termini of the NF proteins along the core fiber can interact with different cytoskeletal components to establish axonal cytoarchitecture
. Structures of the sidearms, which can be altered by NF stoichiometry, influence axonal transport and determines axonal caliber, which affects the conduction velocity of neurons. Therefore, alteration in the NF stoichiometry can contribute to neurodegenerative diseases
 as well as other neurological functions. Up-regulated NF-M protein in the hippocampus of rats fed perilla-diets (Figure
3) can alter the neuronal cell morphology in the hilus of dentate gyrus, a neurogenic area (Figure
5C), thereby facilitating the conduction velocity of neurons to improve learning and memory. Moreover, the up-regulation of neurofilaments in the hilus of dentate gyrus facilitated neurite outgrowth in the perilla-diet groups, suggesting enhanced neurogenesis associated with improved spatial memory performance