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Synaptic plasticity preserved with arachidonic acid diet in aged rats
Neuroscience Research 46 (2003) 453 /461 www.elsevier.com/locate/neures
Synaptic plasticity preserved with arachidonic acid diet in aged rats Susumu Kotani a,b, Hiroe Nakazawa a, Takayuki Tokimasa a,1, Kengo Akimoto c, Hiroshi Kawashima d, Yoshiko Toyoda-Ono c, Yoshinobu Kiso c, Hiroshige Okaichi e, Manabu Sakakibara b,* a
b
Department of Physiology, School of Medicine, Tokai University, Isehara 259-1193, Japan Laboratory of Neurobiological Engineering, School of High-Technology for Human Welfare, Tokai University, 317 Nishino Numazu 410-0321, Shizuoka, Japan c Institute of Health Care Science SUNTORY Ltd, Shimamoto 618-8503, Osaka, Japan d Process Development Department SUNTORY Ltd, Shimamoto 618-8503, Osaka, Japan e Department of Psychology, Doshisha University, Kyoto 602-8580, Japan Received 17 January 2003; accepted 1 April 2003
Abstract We examined whether synaptic plasticity was preserved in aged rats administered an arachidonic acid (AA) containing diet. Young male Fischer-344 rats (2 mo of age), and two groups of aged rats of the same strain (2 y of age) who consumed either a control diet or an AA ethyl ester-containing diet for at least 3 mo were used. In the Morris water maze task, aged rats on the AA diet had tendency to show better performance than aged rats on the control diet. Long-term potentiation induced by tetanic stimulation was recorded from a 300 mm thick hippocampal slice with a 36 multi-electrode-array positioned at the dendrites of CA1 pyramidal neurons. The degree of potentiation after 1 h in aged rats on the AA diet was comparable as that of young controls. Phospholipid analysis revealed that AA and docosahexaenoic acid were the major fatty acids in the hippocampus in aged rats. There was a correlation between the behavioral measure and the changes in excitatory postsynaptic potential slope and between the physiologic measure and the total amount of AA in hippocampus. # 2003 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Aging; Arachidonic acid diet; Long-term potentiation; Water maze test; Lipid analysis; Correlation coefficient; Rat
1. Introduction Long-term potentiation (LTP) is proposed to be a cellular model of synaptic plasticity (Bliss and Lomo, 1973; Kauer et al., 1988). There is general agreement that aged rats exhibit an impaired ability to sustain LTP, but the underlying cause of this deficit is unknown (Landfield et al., 1978; Barnes, 1990; Murray and Lynch, 1998b). Studies of aged animals demonstrated that aging decreased the degree of hippocampal LTP compared with young Fischer-344 rats. Rosenzweig et al. showed
* Corresponding author. Tel.: /81-55-968-1211x4504; fax: /81-55968-1156. E-mail address: [email protected] (M. Sakakibara). 1 Present Address: Kurume rehabilitation Hospital, Kurume 8390827, Fukuoka, Japan.
that LTP-induced deficit was caused by reduction of input cooperativity at the synapse of Schaffer collateral input onto CA1 pyramidal neuron and by impairment of temporal summation of multiple excitatory postsynaptic potentials (EPSPs). These electrophysiologic deficits were in parallel with behavioral deficit of Morris water maze test (Rosenzweig et al., 1997). Recent study by Okada et al. examined the relative importance of the roles of CA1 and dentate gyrus in Morris water maze learning performance, they showed that a lower threshold of LTP induction in CA1 is more crucial for Morris water maze learning than that in the dentate gyrus with manipulation to enhance of synaptic efficacy in CA1 (Okada et al., 2003). In addition, there is an age-dependent decrease in the concentration of arachidonic acid (AA) in the hippocampus (Soderberg et al., 1991; McGahon et al., 1997; Murray and Lynch, 1998b), whereas the uptake of AA
0168-0102/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/S0168-0102(03)00123-8
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into membranes is unchanged (Terracina et al., 1992). This aging-specific effect of AA decline is restored by chronic (8-wk) supplementation of AA or its precursor, g-linolenic acid (McGahon et al., 1997) or recovered by administration of a-tocopherol, antioxidants (Murray and Lynch, 1998a), or chronic treatment with docosahexaenoic acid (DHA), which decreases membrane AA without increasing lipid peroxidation (Treen et al., 1992; Rodriguez et al., 1998; Amamoto et al., 1999). The agedependent suppression of LTP is thought to be a consequence of the AA decline in hippocampal dentate gyrus due to long-term exposure of neurons to oxidative stress (McGahon et al., 1997) or suppression by oxygen free radicals. The relation of LTP and spatial learning ability in aged animals has been examined. Diana et al. suggested that the amplitude of CA1 Ca2-induced LTP in old rats was related to spatial learning ability (Diana et al., 1995). Rosenzweig et al. suggested that there was a deficit in the ability of synapses in aged rats to provide the sustained depolarization necessary to activate the LTP induction cascade (Rosenzweig et al., 1997). In the present study we will demonstrate the characteristics of aged animals supplemented with chronic intake of AA containing food and control food and compare to young rat with control diet from the view point of behavior, electrophysiology and hippocampal fatty acid composition in each identified individual rats.
2. Methods 2.1. Animals and experimental paradigm Male, aged (older than 18 mo) Fischer-344 rats were obtained from Japan Charles River, Yokohama. We kept these rats in the laboratory for 0.5 mo in order to estimate their daily behavior. At the age of 18.5 mo rats were randomly subdivided into two groups; one group (OA) was fed experimental chow supplemented with AA ethyl ester, and the other (OC) was fed control chow for 12 wk. The AA chow contained 2 g AA (SUNTGA40S(ARAVITA 40), SUNTORY, Osaka, Japan) per 1 kg powder chow. The control diet contained no AA, but instead contained additional corn oil (5 g/kg). Animals in both groups were fed 20 g chow per day. Because SUNTGA40S(ARAVITA40) contains AA and the precursor, di-homo-g-linolenic acid (DGLA), daily AA intake for each rat was 40 mg. According to the previous studies Fischer-344 rat begins to die of old age at 80 wk and the life span is know to be 130 wk, we considered rats older than 21 mo were the aged animals (Zanotti et al., 1989; Tayama et al., 1990; Kitani et al., 1994). Behavioral examination was started after at least 21.5 mo. Performance of rats in the Morris water maze was examined at the Laboratory of Psychology at Doshisha
University for 3 wk. After examination of the spatial learning behavior, the animals were transferred to the Neurobiological Engineering Laboratory at Tokai University for electrophysiologic study. Two days after arrival at Tokai University the electrophysiologic study was started at the age of at least 22 mo old. Rats were maintained on a 12-h dark /light schedule (0700 light on, 1900 light off). Ambient temperature was controlled between 23 and 26 8C. Young Fischer-344 rats (2 mo old), obtained from the same supplier were used as controls (YC). Young animals were fed the same diet as the aged control animals. Water and food was freely available for at least 1 mo before performing the electrophysiologic experiments. After electrophysiologic study with a brain slice, the tissue of the hippocampus including each recording slice and the cerebral cortex were frozen at /80 8C for later analysis of fatty acid composition at the Institute of Health Care Science, SUNTORY Ltd. Investigators performing each of the various tests (i.e. behavioral, electrophysiologic, and biochemical) were blind to the results of the other tests. All experiments were performed in accordance with the Doshisha University guidelines and the Tokai University guidelines for animal experiment for behavioral and physiologic study, respectively. 2.2. Water maze Rats were trained for the place task in a shallow pool. A circular polyethylene pool (142 cm in diameter) was placed in the center of the room. There were various objects in the room that served as extramaze visual cues. The height of the wall of the pool was 40 cm, and the depth of the water was 12 cm. The water was made opaque with non-toxic black ink. The escape platform was an 11.5 cm diameter /11 cm high. The rats were required to reach the escape platform from a start position at the rim of the pool. The position of the platform was fixed for each rat, and the two start positions were at the distal ends of the imaginary axes; that is, if the platform was placed in the north /east quadrant, the start positions alternated between the west and south rims of the pool. The rats were trained on the place task with four trials per day for 7 d. A camera mounted on the ceiling above the pool recorded images on a video recorder to analyze escape latency, swimming distance, and swimming path. The water maze test consisted of acquisition (place task) for 7 d and retention test for 2 d. In the present study we exclusively employed two measures of behavior, mean escape latency and hit percent (hit%) other than swimming distance. The escape latency was defined as the time required to find the hidden platform submerged 1 cm. The hit% was obtained from the analysis of swimming path but no further analysis on swimming path was done. The hit% is a the measure of search strategy
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accuracy. An imaginary wedge-shaped area originating from the start point and extending to encompass the area that is 9/30 degrees from the center of the platform was defined as the target region. Hit% was defined as the ratio of time spent in this target region to the total escape latency. This value reflects the animals’ search accuracy to find the platform. The details of the water maze were described previously (Okaichi, 2001). The behavioral data of each day was represented in mean escape latency and mean hit% which were the average value for 4 trials obtained from each individual animal. 2.3. Slice preparation and maintenance Animals were decapitated under full diethyl ether anesthesia. The brain was quickly removed and the hippocampus dissected out in ice-cold working artificial cerebrospinal fluid (Working-ACSF) containing (in mM) 124.0 NaCl, 2.5 KCl, 1.5 CaCl2, 5.0 MgCl2, 1.25 NaH2PO4, 26.0 NaHCO3, and 10.0 glucose (pH :/7.8) and were continually aerated with oxygen, carbon dioxide mixture gas (95% O2 /5% CO2). Transverse slices (300 mm thick) were cut with a vibrating blade microslicer (VT100S, Leica, Heerbrugg, Switzerland). Five or six slices having typical cell layer architecture characterized in hippocampus were obtained from a rat. Slices were kept for more than 30 min at 10 8C in Working-ACSF to reduce metabolic activity and minimize tissue damage. Slices were then transferred and positioned onto a 6 /6 multi electrode dish (MED) on which recordings were made from at least 5 points along a dendrite of a CA1 pyramidal neuron. The slices were submerged under 0.1 /0.2 mm of flowing carboxygenated recording ACSF (same composition as WorkingACSF except 1.5 mM MgCl2 and 2.5 mM CaCl2) at a rate of 2 ml/min and kept at 30 8C. To make close contact with the electrodes slices were stabilized for at least 1 h before starting the experiment. 2.4. Recordings Field recordings from the CA1 region were made with an array of custom-made low-impedance platinum black-coated electrodes (16 /16 mm) embedded in a quartz glass plate, each electrode was separated by 50 mm and the signals were fed into an 8-channel main amplifier (SACC-1, SH-MED8, SU-MED8, Matsushita Electric Co., Osaka, Japan) through a MED probe system (SAGC-5, Matsushita Electric Co.,), and stored in a personal computer using Axoscope (Axon Instruments, Union City, CA). The 6/6 electrode array was alternately used for recording and electrical stimulation. To confirm the electrode position and the neuronal architecture of the pyramidal cell, 1,1?-dioctadecyl3,3,3?,3?-tetramethylindocarbocyanine perchlorate (DiI;
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D-282, Molecular Probes, Eugene, OR) was placed near the axons of the CA1 pyramidal neurons. Field EPSP were induced with a bipolar test stimulus of 9/2 V with a duration of 200 ms generated from an electrical stimulator (DPS-1200D, Dia Medical, Tokyo, Japan) at an electrode along the Schaffer collateral fibers. To confirm the EPSP, paired pulse facilitation was recorded by giving twin test pulses with a 40-ms interval. After 30 min stabilization, control EPSPs were recorded 4 times every 5 min, followed by a tetanic stimulation. Tetanic LTP was elicited by stratum radiatum stimulation with a 1-s 100-Hz volley (pulse duration 500 ms). Following the tetanic stimulation, EPSP was recorded for 60 min at 1 min interval from a slice. One individual EPSP record of a rat was represented by the average value from 4 records of slices in a same rat. These records in OA, OC and YC were analyzed statistically with one-way analysis of variance (ANOVA). If there was a main effect multiple comparison among each group was further done with Fisher’s protected least significant difference. Based on the theoretical consideration that the first derivative of the field EPSP corresponds to the synaptic current (Johnston and Wu, 1995) we analyzed the slope of the field EPSP. The slope of the field EPSP was defined by the maximum slope from the first derivative of the evoked potential. The average slope of the baseline EPSP before tetanus was set as the control. EPSP slopes measured after the induction of LTP were expressed as a ratio to baseline. The analysis performed using macros programming with data analysis software (Origin7, Microcal, Northampton, MA). Physiologic experiments were conducted under blind conditions and the electrophysiologic score was unknown to those handling animals and analyzing the correlations. Regression analysis was performed using Origin7. 2.5. Lipid analysis Lipids in the hippocampus were extracted and purified using the method of Folch et al. (Folch et al., 1957). Purified lipids were separated into neutral lipids, phosphatidylethanolamine, phosphatidylinositol (PI), phosphatidylserine, and phosphatidylcholine, using thinlayer chromatography with silica gel 60 (Merck, Darmstadt, Germany). The solvent system was chloroform/ methanol/acetic acid/water (100/75/7/4, v/v/v/v). The fatty acid residues in the lipid fractions were analyzed according to Sakuradani et al. (Sakuradani et al., 1999). In brief, an internal standard (pentadecanoic acid) was added to each and incubated in methanolic HCl at 50 8C for 3 h to induce transmethylation of fatty acid residues into fatty acid methyl ester. The fatty acid methyl esters were extracted using n -hexane and analyzed with capillary gas /liquid chromatography. Analytical conditions were as follows. Apparatus, Agilent 6890 (Agilent);
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column, SP-2330 (30 m /0.32 mm /0.2 mm, SUPELCO); carrier, He (30 cm/s); column temperature, 180 8C (2 min) followed by /2 8C/min to 220 8C. 2.6. Statistical analysis One-way ANOVA was performed to determine whether there were significant difference between conditions. When ANOVA was used and when this analysis indicated significance (at the 0.05 significance level), Fisher’s Restricted Least Difference was used to determine which conditions were significantly different from each other.
3. Results We evaluated the behavior from 8 animals of each groups. Not all rat was assessed by electrophysilogy because of the accidental happenings during making slices and/or electrophysiologic recording, 5, 5 and 6 rats experienced the water maze test for OC, OA and YC, respectively, were examined. Only the electrophysiologic assessment was done for another 9, 6 and 1 for OC, OA and YC rats, respectively. Consequently 32 rats of 14OC, 11-OA and 7-YC were examined electrophysiologically. After electrophysiologic evaluation, 5 of each rats which were evaluated with both behavioral and physiologic assessment were analyzed their fatty acid composition. The ratio of success to induce LTP was different in three groups; from 7 out of 7 rats, LTP could be induced in YC group, from 9 out of 11 rats in OA group we succeeded in LTP induction, while we could observe LTP from 8 out of 14 in OC group. 3.1. Behavioral analysis Fig. 1 demonstrated behavioral scores with two measures, hit% and escape latency of 8 animals in each group that had 7 training sessions consisting of 4 trials per session for the place task. One-way ANOVA was carried out with these measures. If there was a main effect, multiple comparison with Fisher’s protected least significant difference was done. The ANOVA in hit% among three groups, OA, OC and YC, implied that there was a significant difference in the mean value of hit% with 5% significant level. The same ANOVA for the mean escape latency revealed that there was also a significant difference among OA, OC and YC with 1% significant level. In the place task, the mean hit% and the mean escape latency improved over days (data not shown). The multiple comparison test demonstrated that the significant difference was observed between OC and YC with significant level of 1% in the mean hit% as shown in Fig. 1, while no significant difference was observed between YC and OA. This
Fig. 1. Behavioral measures of the mean hit% (A) and escape latency (B) from 8 samples of OA, OC an YC at the final day of acquisition. These measures were calculated from each individual rat among three groups, OA, OC and YC for 7 consecutive training blocks. Basis on these mean values statistical test was done with one way ANOVA, if there was the main effect the multiple comparison test was done with Fisher’s protected least significant difference. One-way ANOVA revealed that there was the main effect of F (2,21)/4.02, P /0.033 and F (2,21)/12.31, P/0.0003 in the hit% and the escape latency, respectively. Results of multiple comparison test were followed: OC vs. OA P (hit%)/0.074(ns), P (escape latency)/0.97(ns); OC vs. YC P (hit%)/0.005, P (escape latency)/0.00028; OA vs. YC P (hit%)/ 0.134(ns), P (escape latency)/0.00137, ns, not significant. Significant difference (P B/0.01) was observed between OC and YC while there was no significant difference between OA and YC in hit%. On the other hand on escape latency significant difference was observed both between OC and YC, and OA and YC. **, represent a significance level of P B/0.01; ***, represent a significant level of P B/0.001.
demonstrated that there was significant difference between OC and the rest of groups, YC and OA. This implied that the behavior of OA was statistically different from that of OC. In the mean escape latency there was also a significant difference (P B/0.05) both between YC and OC and between OA and YC as shown in Fig. 1. 3.2. Paired pulse facilitation Fig. 2A shows the electrode arrangement with respect to the hippocampal neuronal architecture in the CA1 region. The CA1 pyramidal neuron in Fig. 2A was stained with fluorescent dye, DiI. Each recording
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Paired-pulse facilitation occurs in hippocampal CA1 pyramidal neurons at the homosynapse of excitatory afferents when the same input is stimulated twice in rapid succession (Creager et al., 1980). To confirm that the field EPSP was induced through a homosynaptic input, a twin pulse with a 40-ms interval was applied to the Schaffer collateral pathway. The evoked EPSP of the second component was always greater than the first, e.g. the second EPSP slope was 114.7% as that of the first one in Fig. 2B.
3.3. EPSP slope after long-term potentiation Fig. 3 shows that delivery of a high frequency volley of stimuli to the Schaffer collateral resulted in an immediate increase in the slope of the EPSP in three groups (Fig. 3A). EPSP slope is expressed as a percentage of the slope recorded in the 4 min immediately prior to tetanic stimulation to that recorded at the 60 min after tetanic stimulation. Typical EPSP slope changes from the three experimental groups, YC, OA and OC are shown in Fig. 3A. After a tetanic stimulation at time 0, in the OA group in Fig. 3A the EPSP slope amplitude was potentiated by approximately 200% and was maintained at that level for over 60 min. In the OC group, the EPSP was potentiated by less than 150%. The degree of potentiation in OA was not statistically different with that of the YC. The change in EPSP slope during the last 10 min of the recording in the OA group was significantly larger (P B/0.01) than that of the OC group and there was significant difference between the YC and OC (P /0.0002). No significance was observed between the OA and YC groups (Fig. 3B). This finding suggests that the AA-supplemented diet in aged animals preserved synaptic plasticity to levels similar to young controls. Fig. 2. Electrode arrangement of the MED and the morphology of a dendrite in CA1 pyramidal neuron in hippocampus. A 6/6 array of electrodes were positioned with the electrodes spaced every 50 mm apart. Calibration bar represents 100 mm (A). Paired pulse facilitation in CA1 pyramidal neuron evoked by twin pulses. To confirm that the field EPSP was induced through a homosynaptic input, a twin pulse with a 40-ms interval was applied at the Schaffer collateral. The evoked EPSP of the second component was always greater than that of the first (B).
electrode was positioned along the apical dendrite of a pyramidal neuron. With this electrode arrangement, the field EPSP from at least 5 points within a dendrite in 50 mm steps can be recorded. Synaptic stimulation was applied by electrically stimulating the Schaffer collateral using the two electrodes in the MED that were positioned orthogonal to the recording electrodes and that produced the maximum field EPSP response.
3.4. Phospholipid analysis The volume of total fatty acid linked with phospholipids from the hippocampus of each animal was analyzed. AA and DHA were the major fatty acids in hippocampus and AA was the most prominent. Further, small amounts of DGLA were detected and no EPA was detected. In the hippocampus, there was a non-significant trend for the group of OA to have more AA than the OC group; OA: 3.699/0.76 (n /5), OC: 3.109/0.39 (n /5) mg/g tissue. In the hippocampus, AA was the major unsaturated fatty acid whereas in the cerebrum DHA was the major fatty acid (data not shown). There was more AA-linked with PI than to the other phospholipids (data not shown).
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Fig. 3. Dynamic EPSP slope change in young (YC), age control (OC), and aged arachidonic (OA). Data are presented as 4 min bins of the average values from 1 min interval stimulations. After a tetanic stimulation (indicated with an arrow at the time of 0), the EPSP slope was augmented by 165% and maintained at this level for 60 min in OA while those of OC were potentiated less than 120%. The degree of potentiation in OA was not significantly different with that in YC. Insets show actual evoked responses from typical YC, OC and OA slices recorded just before the tetanic stimulation (indicated by continuous curve) and at 60 min after tetanic stimulation (indicated by dotted curve) (A). The relative EPSP slope change in the three groups was normalized from the data obtained at 60 min after tetanic stimulation (indicated by in A) to the data before tetanus (indicated by ). One-way ANOVA revealed that there was the main effect of F (2,29)/10.165 with significant level of 0.01%. Results of multiple comparison test were followed: OC vs. OA P /0.0082; OC vs. YC P / 0.0002; OA vs. YC P/0.0860(ns), ns, not significant; **, represent a significance level of P B/0.01.
Fig. 4. Correlation of the EPSP slope change vs. hit% (A), and totalAA (mg/g hippocampus) vs. EPSP slope change (B) are shown. Dashed lines represented the linear regression line. Data point in (A) were the average value from 4 slices in a same rat linear regression analysis revealed that EPSP slope change and hit% were highly correlated (correlation coefficient/0.74, P/0.0026, n /14) and total-AA and EPSP slope change had a correlation value of 0.59, P /0.007, n/19. Data points in (A) and (B) were not always corresponded because of no behavioral data or accidental happenings during transportation of brain tissues.
that regression analysis indicated that there was a positive correlation between hit% and electrophysiologic EPSP slope change (R /0.74, P /0.0026, Fig. 4A). There was also a positive correlation between the volume of total-AA and EPSP slope change (R /0.59, P /0.007, Fig. 4B).
4. Discussion 3.5. Regression analysis Regression analysis for three groups (OC, OA and YC) was performed among three parameters; behavior (hit% and escape latency), electrophysiology (EPSP slope change), and levels of total AA. Regression analysis of hit%, AA (total-AA), and EPSP slope change are shown in Fig. 4. Electrophysiologic measures of each data point represented one rat obtained from average value among 4 slices in the same rat. Fig. 4A showed
We examined whether AA was effective in preserving the cognitive ability in aged animals. Each rat was evaluated with four distinctive measures, hit%, escape latency, slope change of LTP and the content of fatty acid in hippocampus. Hit% was the first measure to respond with cognitive judgment for a rat in the place task. It was suggested that the higher the hit% the better the cognitive ability. The statistical results suggested that hit% of OC was significantly lower than that of OA
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and YC between which no significant difference was observed. This implied that aged rats of AA diet demonstrated better performance indirectly in Morris water maze test evaluated with hit% in statistical sense. However we could not rule out that OA was good as YC because there was still a difference which tended to be significant between them. We concluded that AA supplementation seemed to be likely to be effective in preservating the cognitive ability in aged animals compared to aged control animals. Though this study could not reveal the mechanism by which AA acts in the rat hippocampus to preserve spatial learning ability or to rescue LTP maintenance, we demonstrated that AA diet for longer than 3 mo in aged animals preserved learning ability to levels that were the same as young controls in both behavioral and electrophysiologic measures. The degree of potentiation of hippocampal LTP was significantly greater in OA compared with OC. The EPSP slopes were augmented by approximately 165% and maintained for longer than 60 min in OA whereas those of OC were potentiated by less than 120%. Since aging-specific decrease of AA concentration (/20% decrease compared to the young control) in hippocampus of aged rats was observed and this decrease was restored by 8 wk chronic administration of AA to the level of young control revealed by McGahon et al. our data confirmed this tendency but we could not show the significant difference (McGahon et al., 1997). Performance in the Morris water maze is usually evaluated using escape latency and/or the path-length. These two measures are more suitable especially for young rats; they are not adequate at all for use with aged animals that are less active than young animals. During acquisition of learning, the aged animals on the AA diet tended to move toward the escape platform, but did not swim well enough and fast enough to reach the goal due to the deterioration of moving ability. In some instance aged rats stopped swimming during their search for goal and stood there for a rest. That might be why multiple comparison between YC and aged animals resulted in significant difference in the escape latency as shown in Fig. 1. In this case, hit% seemed to be the better measure because it reflected the intention of animals irrespective of their ability to swim. This was clearly demonstrated in the correlation study shown in Fig. 4A. Since the previous study demonstrated that no statistical difference had observed between the young rats of AA supplementation and that of control diet in the LTP slope ratio, we employed the young rats of control diet as the aging control (McGahon et al., 1997). Arachidonic acid is sometimes referred to as a retrograde messenger (Clements et al., 1991; Pellerin and Wolfe, 1991; Duarte et al., 1996). Thus, we could not rule out the possibility that in the OA group chronic administration of AA stimulated liberation of more AA
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from membrane phospholipids onto the presynaptic resulting in the induction and maintenance of greater LTP. Another possibility is that the supplemented AA in the membranes influenced both the pre and postsynaptic membrane through phospholipid metabolic pathways to facilitate mobility of functional protein in the plasma membrane due to increased fluidity. Our present results are consistent with the previous findings by McGahon et al. that dietary supplementation with AA and its precursor g-linolenic acid reversed age-dependent impairment in LTP (McGahon et al., 1997). Though the uptake of AA into membranes was unchanged (Terracina et al., 1992), the relative volume of AA in hippocampus was decreased in aged rats (McGahon et al., 1997; Murray and Lynch, 1998b). This might reflect the age-dependent memory deficit due to oxidative stress to neurons (McGahon et al., 1997) and/ or oxygen free-radical suppression of LTP (Gahtan et al., 1998). The age-related changes in AA concentration linked with phospholipids might be induced by increased production of free radicals. Increases in AAlinked phospholipids are coupled with increased lipid peroxidation and decreased membrane AA concentration (Murray and Lynch, 1998a,b; Murray et al., 1999). Lynch’s group proposed the free radical hypothesis that an age-related increase in lipid peroxidation depletes polyunsaturated fatty acids leading to impairment in hippocampal function and that AA acts as one target for lipid peroxidation in the hippocampus (McGahon et al., 1997; Lynch, 1998). They focused on interleukin-1b (IL1b), a proinflammatory cytokine, as a trigger of lipid peroxidation that resulted in a decrease in the membrane-bound AA concentration, because IL-1b increases lipid peroxidation in aged rat hippocampus (Murray et al., 1999). We cannot determine the mechanism underlying the decreased AA in aged rats from the results of the present study. The possibility of reducing agedependent enzymatic activity such as phospholipase A2, phospholipase A1 or phospholipase C to hydrolyze phospholipids (Lynch and Voss, 1994) should be further investigated. The age-dependent increase in microviscosity in synaptosomal and myelin membranes, reflecting decreased membrane fluidity (Zs-Nagy, 1994), is coupled with impairments in signal transduction mechanisms (Battaini et al., 1995) and age-dependent memory deficits (Gatti et al., 1986; Fonlupt et al., 1994). One consequence of the age-related decreases in AA turnover and concentration is a decrease in membrane fluidity. Because AA has a crucial role as a lipid moiety in membrane phospholipids acting on membrane fluidity, neuronal function is thus modulated indirectly. A theory of aging in which the marked age-dependent changes in membrane composition in relation to lipids has been proposed (Zs-Nagy, 1994). Lipids influence the biophysical characteristics of membranes such as membrane
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fluidity and permeability and thus influence membrane functioning through ion channels and signal transduction. The daily intake of AA diet, 40 mg/d/rat, in the present study, was a reasonable amount for a daily supplement. For future studies, it will be important to determine if the metabolic pathway in aged rats hippocampus supplemented with AA is the same as in young animals.
Acknowledgements This work was supported in part by the ProposedBased New Industry Creative Type Technology R and D Promotion Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan in the field of Biocybernetics (98S18-001-2 to M.S.) and the Regional Innovation Research Development project from the Shizuoka Industrial Creative Organization.
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Gahtan, E., Auerbach, J.M., Groner, Y., Segal, M., 1998. Reversible impairment of long-term potentiation in transgenic Cu/Zn-SOD mice. Eur. J. Neurosci. 10, 538 /544. Gatti, C., Noremberg, K., Brunetti, M., Teolato, S., Calderini, G., Gaiti, A., 1986. Turnover of palmitic and arachidonic acids in the phospholipids from different brain areas of adult and aged rats. Neurochem. Res. 11, 241 /252. Johnston, D., Wu, S., 1995. Foundations of Cellular Neurophysiology. MIT Press, Cambridge. Kauer, J.A., Malenka, R.C., Nicoll, R.A., 1988. NMDA application potentiates synaptic transmission in the hippocampus. Nature 334, 250 /252. Kitani, K., Kanai, S., Carrillo, M.C., Ivy, G.O., 1994. Deprenyl increases the life span as well as activities of superoxide dismutase and catalase but not of glutathione peroxidase in selective brain regions in Fischer rats. Ann. NY Acad. Sci. 717, 60 /71. Landfield, P.W., McGaugh, J.L., Lynch, G., 1978. Impaired synaptic potentiation processes in the hippocampus of aged, memorydeficient rats. Brain Res. 150, 85 /101. Lynch, M.A., 1998. Age-related impairment in long-term potentiation in hippocampus: a role for the cytokine, interleukin-1 beta? Prog. Neurobiol. 56, 571 /589. Lynch, M.A., Voss, K.L., 1994. Membrane arachidonic acid concentration correlates with age and induction of long-term potentiation in the dentate gyrus in the rat. Eur. J. Neurosci. 6, 1008 /1014. McGahon, B., Clements, M.P., Lynch, M.A., 1997. The ability of aged rats to sustain long-term potentiation is restored when the agerelated decrease in membrane arachidonic acid concentration is reversed. Neuroscience 81, 9 /16. Murray, C.A., Clements, M.P., Lynch, M.A., 1999. Interleukin-1 induces lipid peroxidation and membrane changes in rat hippocampus: an age-related study. Gerontology 45, 136 /142. Murray, C.A., Lynch, M.A., 1998a. Dietary supplementation with vitamin E reverses the age-related deficit in long term potentiation in dentate gyrus. J. Biol. Chem. 273, 12161 /12168. Murray, C.A., Lynch, M.A., 1998b. Evidence that increased hippocampal expression of the cytokine interleukin-1 beta is a common trigger for age- and stress-induced impairments in long-term potentiation. J. Neurosci. 18, 2974 /2981. Okada, T., Yamada, N., Tsuzuki, K., Horikawa, H.P., Tanaka, K., Ozawa, S., 2003. Long-term potentiation in the hippocampal CA1 area and dentate gyrus plays different roles in spatial learning. Eur. J. Neurosci. 17, 341 /349. Okaichi, H., 2001. Effects of dorsal-striatum lesion and fimbria-fornix lesion on the problem-solving strategies of rats in a shallow water maze. Cognitive, Affective, Behav. Neurosci. 1, 229 /238. Pellerin, L., Wolfe, L., 1991. Release of arachidonic acid by NMDAreceptor activation in the rat hippocampus. Neurochem. Res. 16, 983 /989. Rodriguez, A., Sarda, P., Nessmann, C., Boulot, P., Leger, C.L., Descomps, B., 1998. Delta6- and delta5-desaturase activities in the human fetal liver: kinetic aspects. J. Lipid Res. 39, 1825 / 1832. Rosenzweig, E.S., Rao, G., McNaughton, B.L., Barnes, C.A., 1997. Role of temporal summation in age-related long-term potentiationinduction deficits. Hippocampus 7, 549 /558. Sakuradani, E., Kobayashi, M., Shimizu, S., 1999. Delta 9-fatty acid desaturase from arachidonic acid-producing fungus. Unique gene sequence and its heterologous expression in a fungus, Aspergillus. Eur. J. Biochem. 260, 208 /216. Soderberg, M., Edlund, C., Kristensson, K., Dallner, G., 1991. Fatty acid composition of brain phospholipids in aging and in Alzheimer’s disease. Lipids 26, 421 /425.
S. Kotani et al. / Neuroscience Research 46 (2003) 453 /461 Tayama, K., Fujii, T., Hiraga, Y., 1990. Comparison of F-344/DuCrj rat and Slc:Wistar-Body weight, survival rate (in Japanese). Fischer rat research working group, Tokyo. Terracina, L., Brunetti, M., Avellini, L., De Medio, G.E., Trovarelli, G., Gaiti, A., 1992. Arachidonic and palmitic acid utilization in aged rat brain areas. Mol. Cell Biochem. 115, 35 /42. Treen, M., Uauy, R.D., Jameson, D.M., Thomas, V.L., Hoffman, D.R., 1992. Effect of docosahexaenoic acid on membrane fluidity
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and function in intact cultured Y-79 retinoblastoma cells. Arch. Biochem. Biophys. 294, 564 /570. Zanotti, A., Valzelli, L., Toffano, G., 1989. Chronic phosphatidylserine treatment improves spatial memory and passive avoidance in aged rats. Psychopharmacology 99, 316 /321. Zs-Nagy, I., 1994. The Membrane Hypothesis of Aging. CRC Press Inc, New York.
Synaptic plasticity preserved with arachidonic acid diet in aged rats Susumu Kotani a,b, Hiroe Nakazawa a, Takayuki Tokimasa a,1, Kengo Akimoto c, Hiroshi Kawashima d, Yoshiko Toyoda-Ono c, Yoshinobu Kiso c, Hiroshige Okaichi e, Manabu Sakakibara b,* a
b
Department of Physiology, School of Medicine, Tokai University, Isehara 259-1193, Japan Laboratory of Neurobiological Engineering, School of High-Technology for Human Welfare, Tokai University, 317 Nishino Numazu 410-0321, Shizuoka, Japan c Institute of Health Care Science SUNTORY Ltd, Shimamoto 618-8503, Osaka, Japan d Process Development Department SUNTORY Ltd, Shimamoto 618-8503, Osaka, Japan e Department of Psychology, Doshisha University, Kyoto 602-8580, Japan Received 17 January 2003; accepted 1 April 2003
Abstract We examined whether synaptic plasticity was preserved in aged rats administered an arachidonic acid (AA) containing diet. Young male Fischer-344 rats (2 mo of age), and two groups of aged rats of the same strain (2 y of age) who consumed either a control diet or an AA ethyl ester-containing diet for at least 3 mo were used. In the Morris water maze task, aged rats on the AA diet had tendency to show better performance than aged rats on the control diet. Long-term potentiation induced by tetanic stimulation was recorded from a 300 mm thick hippocampal slice with a 36 multi-electrode-array positioned at the dendrites of CA1 pyramidal neurons. The degree of potentiation after 1 h in aged rats on the AA diet was comparable as that of young controls. Phospholipid analysis revealed that AA and docosahexaenoic acid were the major fatty acids in the hippocampus in aged rats. There was a correlation between the behavioral measure and the changes in excitatory postsynaptic potential slope and between the physiologic measure and the total amount of AA in hippocampus. # 2003 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Aging; Arachidonic acid diet; Long-term potentiation; Water maze test; Lipid analysis; Correlation coefficient; Rat
1. Introduction Long-term potentiation (LTP) is proposed to be a cellular model of synaptic plasticity (Bliss and Lomo, 1973; Kauer et al., 1988). There is general agreement that aged rats exhibit an impaired ability to sustain LTP, but the underlying cause of this deficit is unknown (Landfield et al., 1978; Barnes, 1990; Murray and Lynch, 1998b). Studies of aged animals demonstrated that aging decreased the degree of hippocampal LTP compared with young Fischer-344 rats. Rosenzweig et al. showed
* Corresponding author. Tel.: /81-55-968-1211x4504; fax: /81-55968-1156. E-mail address: [email protected] (M. Sakakibara). 1 Present Address: Kurume rehabilitation Hospital, Kurume 8390827, Fukuoka, Japan.
that LTP-induced deficit was caused by reduction of input cooperativity at the synapse of Schaffer collateral input onto CA1 pyramidal neuron and by impairment of temporal summation of multiple excitatory postsynaptic potentials (EPSPs). These electrophysiologic deficits were in parallel with behavioral deficit of Morris water maze test (Rosenzweig et al., 1997). Recent study by Okada et al. examined the relative importance of the roles of CA1 and dentate gyrus in Morris water maze learning performance, they showed that a lower threshold of LTP induction in CA1 is more crucial for Morris water maze learning than that in the dentate gyrus with manipulation to enhance of synaptic efficacy in CA1 (Okada et al., 2003). In addition, there is an age-dependent decrease in the concentration of arachidonic acid (AA) in the hippocampus (Soderberg et al., 1991; McGahon et al., 1997; Murray and Lynch, 1998b), whereas the uptake of AA
0168-0102/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/S0168-0102(03)00123-8
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into membranes is unchanged (Terracina et al., 1992). This aging-specific effect of AA decline is restored by chronic (8-wk) supplementation of AA or its precursor, g-linolenic acid (McGahon et al., 1997) or recovered by administration of a-tocopherol, antioxidants (Murray and Lynch, 1998a), or chronic treatment with docosahexaenoic acid (DHA), which decreases membrane AA without increasing lipid peroxidation (Treen et al., 1992; Rodriguez et al., 1998; Amamoto et al., 1999). The agedependent suppression of LTP is thought to be a consequence of the AA decline in hippocampal dentate gyrus due to long-term exposure of neurons to oxidative stress (McGahon et al., 1997) or suppression by oxygen free radicals. The relation of LTP and spatial learning ability in aged animals has been examined. Diana et al. suggested that the amplitude of CA1 Ca2-induced LTP in old rats was related to spatial learning ability (Diana et al., 1995). Rosenzweig et al. suggested that there was a deficit in the ability of synapses in aged rats to provide the sustained depolarization necessary to activate the LTP induction cascade (Rosenzweig et al., 1997). In the present study we will demonstrate the characteristics of aged animals supplemented with chronic intake of AA containing food and control food and compare to young rat with control diet from the view point of behavior, electrophysiology and hippocampal fatty acid composition in each identified individual rats.
2. Methods 2.1. Animals and experimental paradigm Male, aged (older than 18 mo) Fischer-344 rats were obtained from Japan Charles River, Yokohama. We kept these rats in the laboratory for 0.5 mo in order to estimate their daily behavior. At the age of 18.5 mo rats were randomly subdivided into two groups; one group (OA) was fed experimental chow supplemented with AA ethyl ester, and the other (OC) was fed control chow for 12 wk. The AA chow contained 2 g AA (SUNTGA40S(ARAVITA 40), SUNTORY, Osaka, Japan) per 1 kg powder chow. The control diet contained no AA, but instead contained additional corn oil (5 g/kg). Animals in both groups were fed 20 g chow per day. Because SUNTGA40S(ARAVITA40) contains AA and the precursor, di-homo-g-linolenic acid (DGLA), daily AA intake for each rat was 40 mg. According to the previous studies Fischer-344 rat begins to die of old age at 80 wk and the life span is know to be 130 wk, we considered rats older than 21 mo were the aged animals (Zanotti et al., 1989; Tayama et al., 1990; Kitani et al., 1994). Behavioral examination was started after at least 21.5 mo. Performance of rats in the Morris water maze was examined at the Laboratory of Psychology at Doshisha
University for 3 wk. After examination of the spatial learning behavior, the animals were transferred to the Neurobiological Engineering Laboratory at Tokai University for electrophysiologic study. Two days after arrival at Tokai University the electrophysiologic study was started at the age of at least 22 mo old. Rats were maintained on a 12-h dark /light schedule (0700 light on, 1900 light off). Ambient temperature was controlled between 23 and 26 8C. Young Fischer-344 rats (2 mo old), obtained from the same supplier were used as controls (YC). Young animals were fed the same diet as the aged control animals. Water and food was freely available for at least 1 mo before performing the electrophysiologic experiments. After electrophysiologic study with a brain slice, the tissue of the hippocampus including each recording slice and the cerebral cortex were frozen at /80 8C for later analysis of fatty acid composition at the Institute of Health Care Science, SUNTORY Ltd. Investigators performing each of the various tests (i.e. behavioral, electrophysiologic, and biochemical) were blind to the results of the other tests. All experiments were performed in accordance with the Doshisha University guidelines and the Tokai University guidelines for animal experiment for behavioral and physiologic study, respectively. 2.2. Water maze Rats were trained for the place task in a shallow pool. A circular polyethylene pool (142 cm in diameter) was placed in the center of the room. There were various objects in the room that served as extramaze visual cues. The height of the wall of the pool was 40 cm, and the depth of the water was 12 cm. The water was made opaque with non-toxic black ink. The escape platform was an 11.5 cm diameter /11 cm high. The rats were required to reach the escape platform from a start position at the rim of the pool. The position of the platform was fixed for each rat, and the two start positions were at the distal ends of the imaginary axes; that is, if the platform was placed in the north /east quadrant, the start positions alternated between the west and south rims of the pool. The rats were trained on the place task with four trials per day for 7 d. A camera mounted on the ceiling above the pool recorded images on a video recorder to analyze escape latency, swimming distance, and swimming path. The water maze test consisted of acquisition (place task) for 7 d and retention test for 2 d. In the present study we exclusively employed two measures of behavior, mean escape latency and hit percent (hit%) other than swimming distance. The escape latency was defined as the time required to find the hidden platform submerged 1 cm. The hit% was obtained from the analysis of swimming path but no further analysis on swimming path was done. The hit% is a the measure of search strategy
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accuracy. An imaginary wedge-shaped area originating from the start point and extending to encompass the area that is 9/30 degrees from the center of the platform was defined as the target region. Hit% was defined as the ratio of time spent in this target region to the total escape latency. This value reflects the animals’ search accuracy to find the platform. The details of the water maze were described previously (Okaichi, 2001). The behavioral data of each day was represented in mean escape latency and mean hit% which were the average value for 4 trials obtained from each individual animal. 2.3. Slice preparation and maintenance Animals were decapitated under full diethyl ether anesthesia. The brain was quickly removed and the hippocampus dissected out in ice-cold working artificial cerebrospinal fluid (Working-ACSF) containing (in mM) 124.0 NaCl, 2.5 KCl, 1.5 CaCl2, 5.0 MgCl2, 1.25 NaH2PO4, 26.0 NaHCO3, and 10.0 glucose (pH :/7.8) and were continually aerated with oxygen, carbon dioxide mixture gas (95% O2 /5% CO2). Transverse slices (300 mm thick) were cut with a vibrating blade microslicer (VT100S, Leica, Heerbrugg, Switzerland). Five or six slices having typical cell layer architecture characterized in hippocampus were obtained from a rat. Slices were kept for more than 30 min at 10 8C in Working-ACSF to reduce metabolic activity and minimize tissue damage. Slices were then transferred and positioned onto a 6 /6 multi electrode dish (MED) on which recordings were made from at least 5 points along a dendrite of a CA1 pyramidal neuron. The slices were submerged under 0.1 /0.2 mm of flowing carboxygenated recording ACSF (same composition as WorkingACSF except 1.5 mM MgCl2 and 2.5 mM CaCl2) at a rate of 2 ml/min and kept at 30 8C. To make close contact with the electrodes slices were stabilized for at least 1 h before starting the experiment. 2.4. Recordings Field recordings from the CA1 region were made with an array of custom-made low-impedance platinum black-coated electrodes (16 /16 mm) embedded in a quartz glass plate, each electrode was separated by 50 mm and the signals were fed into an 8-channel main amplifier (SACC-1, SH-MED8, SU-MED8, Matsushita Electric Co., Osaka, Japan) through a MED probe system (SAGC-5, Matsushita Electric Co.,), and stored in a personal computer using Axoscope (Axon Instruments, Union City, CA). The 6/6 electrode array was alternately used for recording and electrical stimulation. To confirm the electrode position and the neuronal architecture of the pyramidal cell, 1,1?-dioctadecyl3,3,3?,3?-tetramethylindocarbocyanine perchlorate (DiI;
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D-282, Molecular Probes, Eugene, OR) was placed near the axons of the CA1 pyramidal neurons. Field EPSP were induced with a bipolar test stimulus of 9/2 V with a duration of 200 ms generated from an electrical stimulator (DPS-1200D, Dia Medical, Tokyo, Japan) at an electrode along the Schaffer collateral fibers. To confirm the EPSP, paired pulse facilitation was recorded by giving twin test pulses with a 40-ms interval. After 30 min stabilization, control EPSPs were recorded 4 times every 5 min, followed by a tetanic stimulation. Tetanic LTP was elicited by stratum radiatum stimulation with a 1-s 100-Hz volley (pulse duration 500 ms). Following the tetanic stimulation, EPSP was recorded for 60 min at 1 min interval from a slice. One individual EPSP record of a rat was represented by the average value from 4 records of slices in a same rat. These records in OA, OC and YC were analyzed statistically with one-way analysis of variance (ANOVA). If there was a main effect multiple comparison among each group was further done with Fisher’s protected least significant difference. Based on the theoretical consideration that the first derivative of the field EPSP corresponds to the synaptic current (Johnston and Wu, 1995) we analyzed the slope of the field EPSP. The slope of the field EPSP was defined by the maximum slope from the first derivative of the evoked potential. The average slope of the baseline EPSP before tetanus was set as the control. EPSP slopes measured after the induction of LTP were expressed as a ratio to baseline. The analysis performed using macros programming with data analysis software (Origin7, Microcal, Northampton, MA). Physiologic experiments were conducted under blind conditions and the electrophysiologic score was unknown to those handling animals and analyzing the correlations. Regression analysis was performed using Origin7. 2.5. Lipid analysis Lipids in the hippocampus were extracted and purified using the method of Folch et al. (Folch et al., 1957). Purified lipids were separated into neutral lipids, phosphatidylethanolamine, phosphatidylinositol (PI), phosphatidylserine, and phosphatidylcholine, using thinlayer chromatography with silica gel 60 (Merck, Darmstadt, Germany). The solvent system was chloroform/ methanol/acetic acid/water (100/75/7/4, v/v/v/v). The fatty acid residues in the lipid fractions were analyzed according to Sakuradani et al. (Sakuradani et al., 1999). In brief, an internal standard (pentadecanoic acid) was added to each and incubated in methanolic HCl at 50 8C for 3 h to induce transmethylation of fatty acid residues into fatty acid methyl ester. The fatty acid methyl esters were extracted using n -hexane and analyzed with capillary gas /liquid chromatography. Analytical conditions were as follows. Apparatus, Agilent 6890 (Agilent);
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column, SP-2330 (30 m /0.32 mm /0.2 mm, SUPELCO); carrier, He (30 cm/s); column temperature, 180 8C (2 min) followed by /2 8C/min to 220 8C. 2.6. Statistical analysis One-way ANOVA was performed to determine whether there were significant difference between conditions. When ANOVA was used and when this analysis indicated significance (at the 0.05 significance level), Fisher’s Restricted Least Difference was used to determine which conditions were significantly different from each other.
3. Results We evaluated the behavior from 8 animals of each groups. Not all rat was assessed by electrophysilogy because of the accidental happenings during making slices and/or electrophysiologic recording, 5, 5 and 6 rats experienced the water maze test for OC, OA and YC, respectively, were examined. Only the electrophysiologic assessment was done for another 9, 6 and 1 for OC, OA and YC rats, respectively. Consequently 32 rats of 14OC, 11-OA and 7-YC were examined electrophysiologically. After electrophysiologic evaluation, 5 of each rats which were evaluated with both behavioral and physiologic assessment were analyzed their fatty acid composition. The ratio of success to induce LTP was different in three groups; from 7 out of 7 rats, LTP could be induced in YC group, from 9 out of 11 rats in OA group we succeeded in LTP induction, while we could observe LTP from 8 out of 14 in OC group. 3.1. Behavioral analysis Fig. 1 demonstrated behavioral scores with two measures, hit% and escape latency of 8 animals in each group that had 7 training sessions consisting of 4 trials per session for the place task. One-way ANOVA was carried out with these measures. If there was a main effect, multiple comparison with Fisher’s protected least significant difference was done. The ANOVA in hit% among three groups, OA, OC and YC, implied that there was a significant difference in the mean value of hit% with 5% significant level. The same ANOVA for the mean escape latency revealed that there was also a significant difference among OA, OC and YC with 1% significant level. In the place task, the mean hit% and the mean escape latency improved over days (data not shown). The multiple comparison test demonstrated that the significant difference was observed between OC and YC with significant level of 1% in the mean hit% as shown in Fig. 1, while no significant difference was observed between YC and OA. This
Fig. 1. Behavioral measures of the mean hit% (A) and escape latency (B) from 8 samples of OA, OC an YC at the final day of acquisition. These measures were calculated from each individual rat among three groups, OA, OC and YC for 7 consecutive training blocks. Basis on these mean values statistical test was done with one way ANOVA, if there was the main effect the multiple comparison test was done with Fisher’s protected least significant difference. One-way ANOVA revealed that there was the main effect of F (2,21)/4.02, P /0.033 and F (2,21)/12.31, P/0.0003 in the hit% and the escape latency, respectively. Results of multiple comparison test were followed: OC vs. OA P (hit%)/0.074(ns), P (escape latency)/0.97(ns); OC vs. YC P (hit%)/0.005, P (escape latency)/0.00028; OA vs. YC P (hit%)/ 0.134(ns), P (escape latency)/0.00137, ns, not significant. Significant difference (P B/0.01) was observed between OC and YC while there was no significant difference between OA and YC in hit%. On the other hand on escape latency significant difference was observed both between OC and YC, and OA and YC. **, represent a significance level of P B/0.01; ***, represent a significant level of P B/0.001.
demonstrated that there was significant difference between OC and the rest of groups, YC and OA. This implied that the behavior of OA was statistically different from that of OC. In the mean escape latency there was also a significant difference (P B/0.05) both between YC and OC and between OA and YC as shown in Fig. 1. 3.2. Paired pulse facilitation Fig. 2A shows the electrode arrangement with respect to the hippocampal neuronal architecture in the CA1 region. The CA1 pyramidal neuron in Fig. 2A was stained with fluorescent dye, DiI. Each recording
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Paired-pulse facilitation occurs in hippocampal CA1 pyramidal neurons at the homosynapse of excitatory afferents when the same input is stimulated twice in rapid succession (Creager et al., 1980). To confirm that the field EPSP was induced through a homosynaptic input, a twin pulse with a 40-ms interval was applied to the Schaffer collateral pathway. The evoked EPSP of the second component was always greater than the first, e.g. the second EPSP slope was 114.7% as that of the first one in Fig. 2B.
3.3. EPSP slope after long-term potentiation Fig. 3 shows that delivery of a high frequency volley of stimuli to the Schaffer collateral resulted in an immediate increase in the slope of the EPSP in three groups (Fig. 3A). EPSP slope is expressed as a percentage of the slope recorded in the 4 min immediately prior to tetanic stimulation to that recorded at the 60 min after tetanic stimulation. Typical EPSP slope changes from the three experimental groups, YC, OA and OC are shown in Fig. 3A. After a tetanic stimulation at time 0, in the OA group in Fig. 3A the EPSP slope amplitude was potentiated by approximately 200% and was maintained at that level for over 60 min. In the OC group, the EPSP was potentiated by less than 150%. The degree of potentiation in OA was not statistically different with that of the YC. The change in EPSP slope during the last 10 min of the recording in the OA group was significantly larger (P B/0.01) than that of the OC group and there was significant difference between the YC and OC (P /0.0002). No significance was observed between the OA and YC groups (Fig. 3B). This finding suggests that the AA-supplemented diet in aged animals preserved synaptic plasticity to levels similar to young controls. Fig. 2. Electrode arrangement of the MED and the morphology of a dendrite in CA1 pyramidal neuron in hippocampus. A 6/6 array of electrodes were positioned with the electrodes spaced every 50 mm apart. Calibration bar represents 100 mm (A). Paired pulse facilitation in CA1 pyramidal neuron evoked by twin pulses. To confirm that the field EPSP was induced through a homosynaptic input, a twin pulse with a 40-ms interval was applied at the Schaffer collateral. The evoked EPSP of the second component was always greater than that of the first (B).
electrode was positioned along the apical dendrite of a pyramidal neuron. With this electrode arrangement, the field EPSP from at least 5 points within a dendrite in 50 mm steps can be recorded. Synaptic stimulation was applied by electrically stimulating the Schaffer collateral using the two electrodes in the MED that were positioned orthogonal to the recording electrodes and that produced the maximum field EPSP response.
3.4. Phospholipid analysis The volume of total fatty acid linked with phospholipids from the hippocampus of each animal was analyzed. AA and DHA were the major fatty acids in hippocampus and AA was the most prominent. Further, small amounts of DGLA were detected and no EPA was detected. In the hippocampus, there was a non-significant trend for the group of OA to have more AA than the OC group; OA: 3.699/0.76 (n /5), OC: 3.109/0.39 (n /5) mg/g tissue. In the hippocampus, AA was the major unsaturated fatty acid whereas in the cerebrum DHA was the major fatty acid (data not shown). There was more AA-linked with PI than to the other phospholipids (data not shown).
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Fig. 3. Dynamic EPSP slope change in young (YC), age control (OC), and aged arachidonic (OA). Data are presented as 4 min bins of the average values from 1 min interval stimulations. After a tetanic stimulation (indicated with an arrow at the time of 0), the EPSP slope was augmented by 165% and maintained at this level for 60 min in OA while those of OC were potentiated less than 120%. The degree of potentiation in OA was not significantly different with that in YC. Insets show actual evoked responses from typical YC, OC and OA slices recorded just before the tetanic stimulation (indicated by continuous curve) and at 60 min after tetanic stimulation (indicated by dotted curve) (A). The relative EPSP slope change in the three groups was normalized from the data obtained at 60 min after tetanic stimulation (indicated by in A) to the data before tetanus (indicated by ). One-way ANOVA revealed that there was the main effect of F (2,29)/10.165 with significant level of 0.01%. Results of multiple comparison test were followed: OC vs. OA P /0.0082; OC vs. YC P / 0.0002; OA vs. YC P/0.0860(ns), ns, not significant; **, represent a significance level of P B/0.01.
Fig. 4. Correlation of the EPSP slope change vs. hit% (A), and totalAA (mg/g hippocampus) vs. EPSP slope change (B) are shown. Dashed lines represented the linear regression line. Data point in (A) were the average value from 4 slices in a same rat linear regression analysis revealed that EPSP slope change and hit% were highly correlated (correlation coefficient/0.74, P/0.0026, n /14) and total-AA and EPSP slope change had a correlation value of 0.59, P /0.007, n/19. Data points in (A) and (B) were not always corresponded because of no behavioral data or accidental happenings during transportation of brain tissues.
that regression analysis indicated that there was a positive correlation between hit% and electrophysiologic EPSP slope change (R /0.74, P /0.0026, Fig. 4A). There was also a positive correlation between the volume of total-AA and EPSP slope change (R /0.59, P /0.007, Fig. 4B).
4. Discussion 3.5. Regression analysis Regression analysis for three groups (OC, OA and YC) was performed among three parameters; behavior (hit% and escape latency), electrophysiology (EPSP slope change), and levels of total AA. Regression analysis of hit%, AA (total-AA), and EPSP slope change are shown in Fig. 4. Electrophysiologic measures of each data point represented one rat obtained from average value among 4 slices in the same rat. Fig. 4A showed
We examined whether AA was effective in preserving the cognitive ability in aged animals. Each rat was evaluated with four distinctive measures, hit%, escape latency, slope change of LTP and the content of fatty acid in hippocampus. Hit% was the first measure to respond with cognitive judgment for a rat in the place task. It was suggested that the higher the hit% the better the cognitive ability. The statistical results suggested that hit% of OC was significantly lower than that of OA
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and YC between which no significant difference was observed. This implied that aged rats of AA diet demonstrated better performance indirectly in Morris water maze test evaluated with hit% in statistical sense. However we could not rule out that OA was good as YC because there was still a difference which tended to be significant between them. We concluded that AA supplementation seemed to be likely to be effective in preservating the cognitive ability in aged animals compared to aged control animals. Though this study could not reveal the mechanism by which AA acts in the rat hippocampus to preserve spatial learning ability or to rescue LTP maintenance, we demonstrated that AA diet for longer than 3 mo in aged animals preserved learning ability to levels that were the same as young controls in both behavioral and electrophysiologic measures. The degree of potentiation of hippocampal LTP was significantly greater in OA compared with OC. The EPSP slopes were augmented by approximately 165% and maintained for longer than 60 min in OA whereas those of OC were potentiated by less than 120%. Since aging-specific decrease of AA concentration (/20% decrease compared to the young control) in hippocampus of aged rats was observed and this decrease was restored by 8 wk chronic administration of AA to the level of young control revealed by McGahon et al. our data confirmed this tendency but we could not show the significant difference (McGahon et al., 1997). Performance in the Morris water maze is usually evaluated using escape latency and/or the path-length. These two measures are more suitable especially for young rats; they are not adequate at all for use with aged animals that are less active than young animals. During acquisition of learning, the aged animals on the AA diet tended to move toward the escape platform, but did not swim well enough and fast enough to reach the goal due to the deterioration of moving ability. In some instance aged rats stopped swimming during their search for goal and stood there for a rest. That might be why multiple comparison between YC and aged animals resulted in significant difference in the escape latency as shown in Fig. 1. In this case, hit% seemed to be the better measure because it reflected the intention of animals irrespective of their ability to swim. This was clearly demonstrated in the correlation study shown in Fig. 4A. Since the previous study demonstrated that no statistical difference had observed between the young rats of AA supplementation and that of control diet in the LTP slope ratio, we employed the young rats of control diet as the aging control (McGahon et al., 1997). Arachidonic acid is sometimes referred to as a retrograde messenger (Clements et al., 1991; Pellerin and Wolfe, 1991; Duarte et al., 1996). Thus, we could not rule out the possibility that in the OA group chronic administration of AA stimulated liberation of more AA
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from membrane phospholipids onto the presynaptic resulting in the induction and maintenance of greater LTP. Another possibility is that the supplemented AA in the membranes influenced both the pre and postsynaptic membrane through phospholipid metabolic pathways to facilitate mobility of functional protein in the plasma membrane due to increased fluidity. Our present results are consistent with the previous findings by McGahon et al. that dietary supplementation with AA and its precursor g-linolenic acid reversed age-dependent impairment in LTP (McGahon et al., 1997). Though the uptake of AA into membranes was unchanged (Terracina et al., 1992), the relative volume of AA in hippocampus was decreased in aged rats (McGahon et al., 1997; Murray and Lynch, 1998b). This might reflect the age-dependent memory deficit due to oxidative stress to neurons (McGahon et al., 1997) and/ or oxygen free-radical suppression of LTP (Gahtan et al., 1998). The age-related changes in AA concentration linked with phospholipids might be induced by increased production of free radicals. Increases in AAlinked phospholipids are coupled with increased lipid peroxidation and decreased membrane AA concentration (Murray and Lynch, 1998a,b; Murray et al., 1999). Lynch’s group proposed the free radical hypothesis that an age-related increase in lipid peroxidation depletes polyunsaturated fatty acids leading to impairment in hippocampal function and that AA acts as one target for lipid peroxidation in the hippocampus (McGahon et al., 1997; Lynch, 1998). They focused on interleukin-1b (IL1b), a proinflammatory cytokine, as a trigger of lipid peroxidation that resulted in a decrease in the membrane-bound AA concentration, because IL-1b increases lipid peroxidation in aged rat hippocampus (Murray et al., 1999). We cannot determine the mechanism underlying the decreased AA in aged rats from the results of the present study. The possibility of reducing agedependent enzymatic activity such as phospholipase A2, phospholipase A1 or phospholipase C to hydrolyze phospholipids (Lynch and Voss, 1994) should be further investigated. The age-dependent increase in microviscosity in synaptosomal and myelin membranes, reflecting decreased membrane fluidity (Zs-Nagy, 1994), is coupled with impairments in signal transduction mechanisms (Battaini et al., 1995) and age-dependent memory deficits (Gatti et al., 1986; Fonlupt et al., 1994). One consequence of the age-related decreases in AA turnover and concentration is a decrease in membrane fluidity. Because AA has a crucial role as a lipid moiety in membrane phospholipids acting on membrane fluidity, neuronal function is thus modulated indirectly. A theory of aging in which the marked age-dependent changes in membrane composition in relation to lipids has been proposed (Zs-Nagy, 1994). Lipids influence the biophysical characteristics of membranes such as membrane
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fluidity and permeability and thus influence membrane functioning through ion channels and signal transduction. The daily intake of AA diet, 40 mg/d/rat, in the present study, was a reasonable amount for a daily supplement. For future studies, it will be important to determine if the metabolic pathway in aged rats hippocampus supplemented with AA is the same as in young animals.
Acknowledgements This work was supported in part by the ProposedBased New Industry Creative Type Technology R and D Promotion Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan in the field of Biocybernetics (98S18-001-2 to M.S.) and the Regional Innovation Research Development project from the Shizuoka Industrial Creative Organization.
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