Elsevier

Neurobiology of Aging

Volume 28, Issue 8, August 2007, Pages 1179-1186
Neurobiology of Aging

Arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats

https://doi.org/10.1016/j.neurobiolaging.2006.05.023Get rights and content

Abstract

Previous studies indicate that long-term dietary supplementation with arachidonic acid (AA) in 20-month-old rats (OA) effectively restores performance in a memory task and the induction of long-term potentiation in the hippocampus to the level of young control animals (YC). The present study examined protein mobility using the live cell imaging technique “Fluorescent Recovery After Photobleaching (FRAP)” in YC, old control (OC) and OA neurons in hippocampal slice preparations. Three measures; mobile fraction (Mf), diffusion constant (D) and time constant (τ), were estimated among YC, OC and OA. Each of these parameters was significantly different between OC and YC, suggesting that membrane fluidity is lower in OC than in YC. In contrast, D and τ were comparable in OA and YC, indicating that hippocampal neuronal membranes supplemented with AA were more fluid than those in OC, whereas the fraction of diffusible protein in the bleached region remained smaller than in YC. Long-term administration of AA to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity.

Introduction

Polyunsaturated fatty acids (PUFA) such as arachidonic acid (AA: n-6 PUFA) and docosahexaenoic acid (DHA: n-3 PUFA) have an important role in the induction and maintenance of long-term potentiation (LTP) [6], [9]. Recent behavioral studies suggest a positive correlation between PUFA and cognition in mammals [1], [11], [15], [29], [34]. Previously, we reported that long-term dietary intake (>3 months) of AA in senescent rats older than 21 months improves Morris water–maze performance and the induction and maintenance of hippocampal LTP [19]. Neuronal membranes in the senescent rat hippocampal region have increased microviscosity together with the loss of some receptors, such as muscarinic 1 and NMDA receptors [30], [31], [36]. These age-dependent hippocampal deficits might be restored with long-term dietary intake of AA or l-α-glycerylphosphorylcholine, which protect against membrane oxidation and might facilitate the uptake of cholesterol into the membrane [24], [30].

Experimental studies of changes in hippocampal neural plasticity during the course of aging indicate that there is a shift in the mechanisms that regulate the thresholds for synaptic modification, including Ca2+ channel function and subsequent Ca2+-dependent processes, providing the basis for a model of age-related changes in hippocampal synaptic function [8]. Alterations in the neural membrane phospholipid components not only influence crucial intracellular and intercellular signaling, but also alter many physical properties of the membrane, such as fluidity, phase transition temperature, bilayer thickness and lateral domains [14]. A PUFA deficiency markedly affects neurotransmission, membrane-bound enzyme and ion channel activities and synaptic plasticity [45]. PUFA supplementation in the diet restores membrane fluidity and gene expression, decreases inflammation and modulates neurotransmission [2], [4], [20], [24], [25], [39], [41], [42]. For example, DHA supplementation increases the membrane fluidity of cardiomyocytes [20] and kidney cells [12] and ameliorates the age-related increases in lipid peroxidation and deterioration in fluidity in canalicular plasma membranes [13]. Although n-3 PUFA (e.g., eicosapentaenoic acid and DHA) and n-6 PUFA (e.g., linoleic acid and AA) have contrasting effects on inflammation [2], [4], gene expression [39], [41], [42], proliferation processes [3], platelet membrane fluidity [26], apoptosis [33] and cytoprotective and cytotherapeutic activity [35], PUFA supplementation improves signal transduction processes in association with improvements in learning and memory, peroxisomal disorders and psychotic changes in schizophrenia, depression, hyperactivity, stroke and Alzheimer disease [14], [40].

Several studies of membrane fluidity have assessed membrane microviscosity [17], [30], [46]. In these studies, a membrane suspension obtained from brain tissue homogenate is measured using fluorescence polarization. This method provides information only on the total viscosity of the membrane. The use of green fluorescent protein allows for studies of subcellular localization, mobility, transport routes and binding interactions of proteins in living cells. Live cell imaging, in combination with photobleaching, energy transfer or fluorescent correlation spectroscopy provides many insights into the movement of proteins and their interactions with cellular components. The mobility of a fluorescent protein can be assessed using a specific type of photobleaching technique called fluorescent recovery after photobleaching (FRAP). In this technique, fluorescent molecules in a small region of the cell are irreversibly photobleached using a high-power laser beam and subsequent movement of the surrounding non-bleached fluorescent molecules into the photobleached area is recorded with lower laser power. Two typical kinetic parameters of a protein can be discerned from quantitative studies using FRAP: the mobile fraction, Mf, which is the fraction of fluorescent proteins that can diffuse into the bleached region during the time course of the experiment and the diffusion constant, D, which is a measure of the rate of protein movement in the absence of low or active transport [10], [16], [22].

Kessler and Yehuda reported a marked decrease in learning-induced cholesterol levels in the hippocampus and a learning-induced increase in membrane lipid fluidity. They postulated that upon learning, brain membranes undergo changes in the lipid membrane phase that facilitate the transduction of the learning process into a biochemical template [17]. The number of muscarinic receptors in the hippocampus of aged rats is significantly lower than that in young animals [30]. Treatment of aged rats with S-adenosyl-l-methionine, however, restores the number of muscarinic receptors [31]. Though the dissociation constants are not different among young and S-adenosyl-l-methionine-treated and-untreated aged rats, binding capacity varies. The reduction in the muscarinic receptor density might be related to the aging-induced decrease in neuronal membrane fluidity [31]. Scheuer et al. demonstrated that piracetam alters membrane fluidity in the hippocampus and that the effects of piracetam on NMDA density in the hippocampus might be involved in its positive effects on cognitive performance [36]. Chronic treatment of aged rats with l-α-glycerylphosphorylcholine restores the number of muscarinic 1 receptors and partially restores membrane fluidity [30].

Cotrina suggested that astrocytic gap junction coupling in mouse hippocampus tends to be reduced as animals age. Using the FRAP technique, fluorescence recovery after 2 min was 63 ± 6% in younger animals, 59 ± 5% in adult animals and 54 ± 4% in old brain. Their observations indicate that although astrocytic gap junction proteins are maintained at high levels throughout the lifespan of the animal, aging is associated with changes in the number and size of both Cx30 and Cx43 gap junction plaques [5].

In the present study, we used FRAP to evaluate and compare membrane fluidity of hippocampal pyramidal neurons among young, aged control and aged animals given long-term dietary supplementation with AA.

Section snippets

Animals

All experiments were performed with Fischer 344 male rats 2 months of age (young control, YC) or 18 months of age (aged animals) obtained from Clea Japan, Tokyo. Aged rats were subdivided into two groups; one group (old animals, OA) was fed experimental chow supplemented with AA containing triglyceride and the other (old control, OC) was fed control chow for 12 weeks. The AA chow contained 2 g AA [SUNTGA40-S (ARAVITA 40), SUNTORY, Osaka, Japan] per kilogram powder chow. The control diet

Results

In the present study, the number of animals used was 3 YC, 2 OC and 2 OA. Five or six hippocampal slices were obtained from each rat. The degree of staining with DiI C16(3) at the soma of the CA1 pyramidal neurons among the three groups was bright enough to detect the recovery of the fluorescent intensity based on microscopic observation. Because we did not measure the absolute intensity level of the photobleached area of each group, it was difficult to compare the staining level among YC, OC

Discussion

We used FRAP to demonstrate that there was a significant difference in membrane fluidity in hippocampal neurons between YC and OC; hippocampal pyramidal neurons from young animals were more fluid, i.e., had more lateral mobility, than those of senescent animals, as characterized by a larger diffusion constant and mobile fraction and a smaller time constant. Long-term (>3 months) AA supplementation kept the hippocampal neuronal membranes more fluid, but the amount of protein molecules available

Acknowledgements

We thank Mr. Shingo Maeda of Olympus Co. for technical assistance. This work was supported by grants from Tokai University for the Promotion of Scientific Research and Suntory Ltd. to M.S.

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