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. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: Nutr Res. 2010 Oct;30(10):722–730. doi: 10.1016/j.nutres.2010.09.008

Short-term nutritional folate deficiency in rats has a greater effect on choline and acetylcholine metabolism in the peripheral nervous system than in the brain, and this effect escalates with age

Natalia A Crivello 1,2,*, Jan K Blusztajn 3, James A Joseph 4, Barbara Shukitt-Hale 4, Donald E Smith 5
PMCID: PMC3000554  NIHMSID: NIHMS238859  PMID: 21056288

Abstract

The hypothesis of this study is that a folate-deficient diet (FD) has a greater effect on cholinergic system in the peripheral nervous system than in the brain, and that this effect escalates with age. It was tested by comparing choline and acetylcholine levels in male Sprague Dawley rats fed either control or folate-deficient diets for 10 weeks, starting at age 4 weeks (the young group) or 9 months (the adult group). FD consumption resulted in depletion of plasma folate in both age groups. In young folate-deficient rats, liver and lung choline levels were significantly lower than those in the respective controls. No other significant effects of FD on choline and acetylcholine metabolism were found in young rats. In adult rats, FD consumption markedly decreased choline levels in the liver, kidneys, and heart; furthermore, choline levels in the cortex and striatum were moderately elevated, although hippocampal choline levels were not affected. Acetylcholine levels were higher in the heart, cortex, and striatum but lower in the hippocampus in adult folate-deficient rats, as compared to controls. Higher acetylcholine levels in the striatum in adult folate-deficient rats were also associated with higher dopamine release in the striatal slices. Thus, both age groups showed higher cholinergic metabolic sensitivity to FD in the peripheral nervous system than in the brain. However, compensatory abilities appeared to be better in the young group, implicating the adult group as a preferred model for further investigation of folate-choline-acetylcholine interactions and their role in brain plasticity and cognitive functions.

Keywords: Folic acid, choline, neurotransmitters, liver, hippocampal choline, rats

1. Introduction

Folate, a cofactor in one-carbon metabolism, is intimately involved in the methylation of homocysteine to methionine and S-adenosylmethionine, and it is one of the most important methyl-group donors in mammals [1–5]. Methyl-group donors are required for many methylation reactions involved in phospholipid, DNA, protein, and neurotransmitter synthesis [2, 5–10]. Folate is metabolically connected to choline, another important methyl-group donor, because the synthesis of methionine from homocysteine can be accomplished by either of two enzymes: 5-methyltetrahydrofolate-homocysteine methyltransferase (which requires vitamin B12 and uses methyltetrahydrofolate as a methyl donor) or by betaine:homocysteine S-methyltransferase (which uses betaine, an oxidized form of choline, as a methyl donor). Choline is also utilized for the synthesis of a neurotransmitter (acetylcholine), for the synthesis of cell-membrane components (phospholipids), and for lipid transport (phospholipids within lipoproteins) [3, 11–15]. The mechanisms by which choline metabolism is regulated have received much attention because the availability of choline can influence the synthesis and release of acetylcholine, which is involved in learning and short-term memory in animals and humans [16, 17]. The correlation of clinical dementia ratings with reductions in a number of cholinergic markers, including acetylcholine levels, suggests an association between cholinergic hypofunction and cognitive deficits [18–20]. Dysregulation of the cholinergic system has been increasingly recognized as an important determinant of both cognitive decline in brain aging and age-related neurodegeneration [3, 4, 20–22]. Because the capacity of the adult brain to synthesize choline de novo is very low, the brain is dependent on the uptake of choline from the blood [23–27].

Both animal [4, 28–33] and human [3, 34–36] studies emphasize the important role of choline for physiologic integrity when sufficient levels of folate or methionine are not available in the diet. Choline- or choline-methionine-deficient diets induce severe depletion of hepatic folate in rats, suggesting that choline deficiency leads to increased utilization of folate to maintain hepatic methionine and S-adenosylmethionine [28–30, 37]. Folate deficiency has recently been reported to reduce brain acetylcholine levels and cause abnormalities of synaptic function in vitro in mice that lack apolipoprotein E [33, 38]. The metabolic relationships among folate, choline, and acetylcholine have been increasingly recognized as important because of the roles of these compounds in brain plasticity and behavior [2–4, 21, 39, 40]. Furthermore, recent evidence has suggested that a full understanding of the metabolic relationships between these three compounds necessitates the investigation of their metabolism in the brain and peripheral tissues.

The present study was designed to test the hypothesis that a folate deficient diet has a greater effect on choline and acetylcholine metabolism in the peripheral nervous system than in the brain, and that this effect escalates with age. The objectives for the study were as follows: (1) to analyze and compare changes in the choline and acetylcholine levels in the brain and peripheral tissues in male Sprague Dawley rats that were fed either a control or folate deficient diets for 10 weeks; (2) determine whether there are differences in choline and acetylcholine metabolisms in response to folate deficiency between young (4 weeks old) and adult (9 months old) rats.

Folate deficiency is one of the most significant dietary health problems worldwide [1]. Poor folate status affects approximately 5–10% of the population exposed to folate fortification in food and more than 30% of the population without such fortification. Therefore, identifying the mechanisms that are associated with folate deficiency will be essential for targeting modifiable risk factors for cognitive decline in the elderly population.

2. Methods and materials

2.1. Materials

Reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and Fisher Scientific (Houston, TX, USA). Diet constituents, including basal mix (99%, AIN 93M) (cat# TD.09171), vitamin mix AIN-93-VX (cat# TD.94047) and folate deficient vitamin mix (cat# TD.95052), were purchased from Harlan Laboratories, Inc. (Madison, WI, USA). Standards for choline (cat# CF- 1042) and acetylcholine (cat# CF-1043) were obtained from Bioanalytical Systems, Inc. (Indiana, USA). Folate standard was obtained from Sigma Aldrich (cat# F7876). Dopamine standard was purchased from Sigma (cat# H8502).

2.2. Diets

Control and folate deficient diets were prepared in the JM USDA HNRCA animal diet preparation kitchen by mixing 99% basal mix with 1% of an appropriate vitamin mix as previously described [31, 32]. Basal mix (cat# TD.09171) contained 1% succinylsulfathiazole, a non-absorbed sulfa drug that inhibits folate formation by gut bacteria, to ensure that the animal’s only source of available folate was from its diet. Diets were formulated with vitamin-free, ethanol-precipitated casein and the appropriate vitamin-mix. Diets were stored at 4°C until used. The compositions of the diets are summarized in Table 1.

Table 1.

Composition of diets fed to rats for 10 weeksa

Dietary treatments
Ingredients (g/kg diet) 99% Basal mixb Controlc Folate deficient
Casein 141.41 140.00 140.00
L-Cystine 1.82 1.80 1.80
Corn Starch 460.29 455.69 455.69
Maltodextrin 156.57 155.00 155.00
Sucrose 101.01 100.00 100.00
Soybean Oil 40.40 40.00 40.00
Cellulose 50.51 50.00 50.00
AIN-93M, mineral mix 35.35 35.00 35.00
Choline bitartrate 2.53 2.50 2.50
TBHQ, antioxidant 0.01 0.01 0.01
Succinylsulfathiazole 10.10 10.00 10.00
Folate sufficient vitamin mixd - 10.00 -
Folate deficient vitamin mixe - - 10.00
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a

Diets were prepared in the JM USDA HNRCA animal diet preparation kitchen by mixing 99%basal mix with 1% of an appropriate vitamin mix as previously described [31, 32]. Dietary ingredients were supplied by Harlan Laboratories, Inc. (Madison, WI, USA).

b

TD.09171 = basal mix (AIN-93M mix without vitamin mix).

c

Folate sufficient diet.

d

TD.94047 = Folate sufficient vitamin mix (AIN-93 vitamin mix with folic acid).

e

TD.95052 = Folate deficient vitamin mix (AIN-93 vitamin mix without folic acid).

2.3. Animals

Young (4 weeks old, n = 10) and adult (9 months old, n = 12) male Sprague Dawley rats were obtained from Charles River Laboratories. All experiments were approved by the Animal Care and Use Committee of the Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University (Boston, MA). Animals were provided with water ad libitum and AIN-93 diet during a one-week acclimatization period, then randomly divided into two groups (young, n = 5 per group; adult, n = 6 per group) and maintained on the experimental diets for 10 weeks. Power analyses indicated that we required 5 rats per group to obtain an 80% chance at the 0.05 level of significance of detecting a difference in means of 196 pmol dopamine/mg protein, 20 nmol choline/mg tissue, and 0.7 nmol acetylcholine/mg tissue. In order to ensure comparable food intake by all groups, animals were group pair-fed. The amount of food provided to each animal was adjusted daily to ensure comparable rates of intake within age and diet groups. This procedure ensured that all age-matched animals had comparable daily consumptions of calories and nutrients. Body mass was recorded weekly. Notable observations, including all those relating to animal health, were also recorded.

2.4. Sample collection

After completion of the ten-week feeding period, the rats were fasting overnight, weighed and euthanized by decapitation. Trunk blood was collected into Vacutainer tubes (Becton Dickinson, Rutherford, NJ) containing 0.1% EDTA. Plasma samples were separated at 2500 rpm for 15 minutes at 4°C then immediately aliquoted and stored frozen at −80°C for future analyses. After blood collection, the cortex, hippocampus, striatum, liver, kidneys, heart, and lungs of each animal were rapidly removed and weighed. The striatum from randomly chosen individual animals were used for the immediate assessment of dopamine release; the remaining striatum and other tissues were snap-frozen in liquid nitrogen. Frozen tissues were ground to a fine powder in liquid nitrogen. Tissue powder was aliquoted and stored at −80°C until further biochemical analyses.

2.5. Blood analyses

Plasma samples (100 µL) were analyzed for glucose, albumin, total cholesterol, HDL, triglycerides, AST, ALT, and creatinine levels using an automated clinical biochemical analyzer, Olympus AU400 (Olympus, TX, USA). All parameters were measured using Olympus reagents. Plasma folate levels were analyzed using standard competitive immunochemiluminometric method for IMMULITE 1000 analyzer (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA). Plasma choline levels were measured using HPLC system coupled to electrochemical detection as described in section 2.5.

2.6. Acetylcholine and choline analyses

Choline and acetylcholine levels were analyzed in plasma and tissue samples using HPLC system coupled to electrochemical detection as described previously [41]. Briefly, choline and acetylcholine were extracted from plasma (100 µL) and tissue (80–100 mg) samples using methanol – 1M formic acid – chloroform - water (1:0.1:2:1 by volume) solution. The supernatant fraction was collected after centrifugation at 2500 rpm for 15 minutes at 4°C, dried under a vacuum, and reconstituted in water. The content of acetylcholine and choline was measured using a Bioanalytical System, Inc (West Lafayette, IN, USA) commercial kit consisting of acetylcholine/choline analytical column and acetylcholinesterase/choline oxidase immobilized enzyme reactor column on HPLC system coupled to electrochemical detector. Data were expressed in µmol/L for plasma samples and in nmol/g for tissue samples.

2.7. Dopamine release analyses

Dopamine release in striatal slices was assessed using HPLC system coupled to electrochemical detection as described previously [42]. Briefly, cross cut (300 µm, McIlwain tissue chopper) slices were equilibrated in low KCl basal release buffer (21 mM NaHCO3, 3.4 mM glucose, 1.3 mM NaH2PO4, 1 mM EGTA, 0.93 mM MgCl2, 127 mM NaCl and 2.5 mM KCl; pH 7.4) for 30 minutes at 37°C. Following a 30-min equilibration period, the buffer was replaced with a high KCL buffer (30 mM KCl, 1.26 mM CaCl2x2H2O, 57 mM NaCl) in order to depolarize the neurons and evoke dopamine release, which was stimulated by 0 or 500 µM of oxotremorine, a non-selective muscarinic acetylcholine receptor agonist. Samples were collected and analyzed using HPLC system (Bioanalytical System, Inc., West Lafayette, IN, USA) coupled to electrochemical detection as described previously [42]. The HPLC system consisted of reverse phase (C18) column (Bioanalytical System, Inc., West Lafayette, IN, USA). The mobile phase was 100 mM KH2PO4 buffer containing 3 mM 1-heptane-sulforic acid, 100 µM EDTA, and 5.5% acetonitrile (pH 3.6) at a flow rate of 1 mL/min. Data were expressed as picomoles per milligram of protein.

2.8. Statistical analyses

The data were analyzed using two-way analyses of variance with age group and diet as study factors using Systat version 10.0 (SPSS, Inc. Chicago, IL). The effect of folate deficiency was judged different for young and adult rats, if the P value for the age vs diet interaction was less than 0.05. If the interaction P value was less than 0.05 Fisher least-significant-difference tests were performed [43]. All values were expressed as means ± SEM. Later in the text we will refer to the young vs. adult effect as the “age effect” and the control diet vs. folate deficient diet effect as the “diet effect.”

3. Results

All experimental animals completed the study without any apparent health complications. The rats tolerated the diets well, with a steady increase in weight over the 10-wk period. A significant difference in body mass (P < .05) between young and adult rats were observed, regardless of dietary folate status (Table 2). There were significant increases in cerebral cortex (P < .01) kidney (P < .01), and heart (P < .05) mass in adult rats as compared to those in young animals. Decreases in the ratio between tissue and whole body mass were observed in the liver (P < .01) and kidney (P < .001) in adult rats, whereas folate deficiency increased kidney-body ratio in both age groups (P < .05) (Table 2).

Table 2.

Body and tissues mass of young and adult rats fed either control or folate-deficient diets

Dietary treatments
Units Control (n = 5) Folate deficient (n = 6)
Body mass (young rats) g 397 ± 70a 364 ± 10b
Body mass (adult rats) g 562 ± 21a 566 ± 17b
Cortex (young rats) mg 487 ± 41c 561 ± 103d
Cortex (adult rats) mg 812 ± 83c 768 ± 42d
Hippocampus (young rats) mg 49 ± 10 46 ± 5
Hippocampus (adult rats) mg 43 ± 6 56 ± 6
Striatum (young rats) mg 24 ± 3 17 ± 2
Striatum (adult rats) mg 25 ± 5 28 ± 7
Liver (young rats) mg 5865 ± 499 5977 ± 152
Liver (adult rats) mg 5739 ± 91 5728 ± 43
Kidney (young rats) mg 1940 ± 127 2031 ± 67e
Kidney (adult rats) mg 2233 ± 55 2518 ± 13e
Heart (young rats) mg 1355 ± 277 971 ± 56 f
Heart (adult rats) mg 1591 ± 192 1456 ± 34f
Lungs (young rats) mg 1470 ± 349 1295 ± 109
Lungs (adult rats) mg 1817 ± 118 2033 ± 418
Cortex-Body ratio (young rats) mg/g 1.22 ± 0.09 1.52 ± 0.25
Cortex-Body ratio (adult rats) mg/g 1.44 ± 0.13 1.36 ± 0.07
Hippocampus-Body ratio (young rats) mg/g 0.12 ± 0.03 0.13 ± 0.02
Hippocampus-Body ratio (adult rats) mg/g 0.08 ± 0.01 0.10 ± 0.01
Striatum -Body ratio (young rats) mg/g 0.06 ± 0.01 0.04 ± 0.01
Striatum -Body ratio (adult rats) mg/g 0.05 ± 0.01 0.05 ± 0.01
Liver-Body ratio (young rats) mg/g 14.72 ± 1.11g 16.44 ± 0.21h
Liver-Body ratio (adult rats) mg/g 10.28 ± 0.49g 10.18 ± 0.36h
Kidney-Body ratio (young rats) mg/g 4.88 ± 0.28 i,k 5.59 ± 0.13j,k
Kidney-Body ratio (adult rats) mg/g 4.01 ± 0.24i 4.45 ± 0.19j
Heart-Body ratio (young rats) mg/g 3.35 ± 0.80 2.67 ± 0.15
Heart-Body ratio (adult rats) mg/g 2.82 ± 0.28 2.59 ± 0.11
Lungs-Body ratio (young rats) mg/g 3.71 ± 0.88 3.56 ± 0.29
Lungs-Body ratio (adult rats) mg/g 3.27 ± 0.29 3.56 ± 0.66
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Data are means ± SEM. Values in the same column or row that share the same superscript letter are significantly different (analysis of variance, P < .05).

3.1. Plasma biochemistry

There were age (P < .001), diet (P < .001), and age by diet (P < .001) effects on plasma folate (Table 3). Folate depletion was observed in both age groups. Plasma folate levels were significantly lower (∼50%) in adult rats as compared to young animals fed a control diet. However, folate levels were not different between the two age groups fed a folate deficient diet (Table 3). Similar to plasma folate, choline levels were different between the two age groups (P < .01) and were further affected by folate deficiency (P < .01). Choline levels were not affected by folate deficiency in young rats, whereas in adult rats plasma choline was approximately 50% higher in the folate deficient group as compared to the respective control group (Table 3). Adult rats fed a control diet showed higher glucose (P < .01) and lower albumin (P < .001) levels in plasma as compared to those in the young group. Folate deficiency significantly increased plasma glucose and albumin levels in young rats but did not affect either glucose or albumin levels in adult rats. Total cholesterol levels were increased (∼30%) in the older rats regardless of diet (P < .05) (Table 3). Biochemical markers of liver (alanine aminotransferase, ALT; and aspartate aminotransferase, AST) and kidney (blood creatinine) functions were not affected by folate deficiency regardless of age (Table 3).

Table 3.

The effect of folate deficiency on plasma biochemistry in young and adult rats

Measurements Dietary treatments
Units Control (n = 5) Folate deficient (n = 6)
Folate (young rats) nmol/L 136.0 ± 11.0a,f 2.9 ± 0.4a
Folate (adult rats) nmol/L 71.0 ± 5.0b,f 5.0 ± 0.7b
Choline (young rats) µmol/L 21.0 ± 2.0 16.0 ± 2.0g
Choline (adult rats) µmol/L 23.0 ± 2.0c 36.0 ± 2.0c,g
Glucose (young rats) mmol/L 6.5 ± 0.2d,h 8.2 ± 0.4d
Glucose (adult rats) mmol/L 7.8 ± 0.2h 7.7 ± 0.2
Albumin (young rats) g/L 35.0 ± 0.2e,i 37.0 ± 0.6e,j
Albumin (adult rats) g/L 32.0 ± 0.1i 33.0 ± 0.3j
Total cholesterol (young rats) mmol/L 2.6 ± 0.3k 2.2 ± 0.2
Total cholesterol (adult rats) mmol/L 3.0 ± 0.1k 2.4 ± 0.1
HDL (young rats) mmol/L 0.9 ± 0.1 0.8 ± 0.1
HDL (adult rats) mmol/L 1.0 ± 0.1 0.8 ± 0.1
Triglycerides (young rats) mmol/L 1.0 ± 0.2 0.6 ± 0.2
Triglycerides (adult rats) mmol/L 1.1 ± 0.1 1.0 ± 0.1
ALT (young rats) U/L 62.0 ± 7.0 64.0 ± 7.0l
ALT (adult rats) U/L 61.0 ± 7.0 48.0 ± 5.0l
AST (young rats) U/L 349.0 ± 49.0 323.0 ± 55.0
AST (adult rats) U/L 243.0 ± 17.0 170.0 ± 19.0
Creatinine (young rats) µmol/L 33.7 ± 1.2 29.2 ± 1.1m
Creatinine (adult rats) µmol/L 33.1 ± 2.0 30.2 ± 1.1m
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Data are means ± SEM. Values in the same column or row that share the same superscript letter are significantly different (analysis of variance, P < .05).

3.2. Tissue choline

Choline metabolism was significantly affected by folate status and by aging, as reflected in the tissue-specific choline content (Table 4). Folate deficiency effects on choline were observed in the liver (P < .001), kidney (P < .001), lungs (P < .01), and heart (P < .01); age effects were recorded in the liver (P < .01), kidney (P < .01), lungs (P < .001), cortex (P < .001), hippocampus (P < .001), and striatum (P < .001) (Table 4). There was a significant age by diet effect on choline levels in the striatum (P < .01), kidney (P < .001), and lungs (P < .001). Folate deficiency resulted in significant decrease in choline levels in the liver and lungs of young rats (40% and 50%, respectively). Choline levels in the peripheral tissues of adult rats were even more affected by folate deficiency. Specifically, there was a significant depletion of choline in the liver (by 70%), kidney (by 50%), and heart (by 50%), whereas in the cortex and striatum, choline levels were increased as compared to those in the respective control (by 30% and 20%, respectively) (Table 4).

Table 4.

The effect of folate deficiency on tissue choline levels in young and adult rats

Dietary treatments
Choline levels (nmol/g) Control (n = 5) Folate deficient (n = 6)
Cortex (young rats) 217 ± 19a 214 ± 20b
Cortex (adult rats) 312 ± 36a,h 404 ± 18b,h
Hippocampus (young rats) 91 ± 10 84 ± 2c
Hippocampus (adult rats) 114 ± 8 135 ± 10c
Striatum (young rats) 523 ± 25d,i 451 ± 22i
Striatum (adult rats) 325 ± 29d,j 403 ± 13j
Liver (young rats) 277 ± 32k 176 ± 2e,k
Liver (adult rats) 243 ± 15l 83 ± 4e,l
Kidneys (young rats) 428 ± 20f 421 ± 12
Kidneys (adult rats) 612±34f,m 399 ± 2m
Lungs (young rats) 561 ± 49g,n 289 ± 22n
Lungs (adult rats) 213 ± 31g 284 ± 20
Heart (young rats) 86 ± 6 69 ± 4
Heart (adult rats) 79 ± 9o 57 ± 5o
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Data are means ± SEM. Values in the same column or row that share the same superscript letter are significantly different (analysis of variance, P < .05).

3.3. Acetylcholine and dopamine

An age effect on acetylcholine was recorded in the cortex (P < .001) and heart (P < .001), whereas a folate deficiency effect was observed in the cortex (P < .05), striatum (P < .001), and heart (P < .01) (Table 5). There was a significant age by diet effect on acetylcholine in the striatum (P < .001) and heart (P < .05) (Table 5). Almost a 50% increase in acetylcholine levels was observed in the cortex and heart of adult rats fed a control diet as compared to levels in young rats within the same dietary group (Table 5). These increases were further potentiated by the exposure of the adult rats to a folate deficient diet. Age-related decreases in striatal acetylcholine were reversed by exposure of rats to a folate deficient diet (Table 5). High levels of acetylcholine in the striatum of adult rats fed a folate deficient diet were associated with significant changes in dopamine release in the striatal slices. Specifically, there was a significant diet effect on dopamine release in the striatum (P < .01) (Fig. 1). Dopamine release in young rats was not affected by folate deficiency. However, there was a significant increase in dopamine release in adult rats fed a folate deficient diet as compared to those fed a control diet (P < .01) (Fig. 1).

Table 5.

The effect of folate deficiency on tissue acetylcholine levels in young and adult rats

Dietary treatments
Acetylcholine levels (nmol/g) Control (n = 5) Folate deficient (n = 6)
Cortex (young rats) 2.42 ± 0.39a 2.95 ± 0.35b
Cortex (adult rats) 4.19 ± 0.50a,h 6.25 ± 0.67b,h
Hippocampus (young rats) 1.40 ± 0.13c 1.74 ± 0.29
Hippocampus (adult rats) 2.11 ± 0.08c,i 1.50 ± 0.09i
Striatum (young rats) 2.89 ± 0.2d 3.25 ± 0.3e
Striatum (adult rats) 1.42 ± 0.10d,j 4.51 ± 0.20e,j
Heart (young rats) 0.97 ± 0.05f 1.17 ± 0.13g
Heart (adult rats) 1.82 ± 0.23f,k 3.14 ± 0.34g,k
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Data are means ± SEM. Values in the same column or row that share the same superscript letter are significantly different (analysis of variance, P < .05).

Fig. 1.

Fig. 1

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The effect of folate deficiency on dopamine release was assessed in in vitro striatal slices in young and adult rats fed either control or folate-deficient diets as described previously [45], The assay measures dopamine release at 0 µM or 500 µM oxotremorine (non-stimulated and stimulated dopamine release, respectively). The results expressed as delta between stimulated and non-stimulated values (delta) Data are mean ± SEM. Asterisk denotes P<.05 (analysis of variance).

4. Discussion

The present study shows that folate deprivation results in significant depletion of plasma folate in rats regardless of age. The data are consistent with our previous report of folate deficiency in young rats [31, 32]. There were no visible health issues in experimental animals regardless of age. However, results show significant differences between the two age groups in adaptation to folate deficiency as reflected by changes in choline and neurotransmitter metabolism in brain and peripheral tissues. Young rats showed more efficient adaptive responses to folate deprivation. Specifically, choline depletion was recorded only in the liver and lungs in young rats fed a folate deficient diet. No other effects of folate deficiency on cholinergic neurotransmitters and tissue choline levels were recorded in young rats.

Conversely, there were significant changes in choline and acetylcholine levels and of dopamine release in adult rats exposed to folate deprivation. The depletion of plasma folate in adult rats fed a folate deficient diet was associated with depletion of choline in the liver, kidney, and heart. Furthermore, adult rats showed greater choline depletion in the liver under folate deficiency than young rats. Earlier studies in young rats demonstrated a similar depletion of hepatic choline under folate deprivation, suggesting that folate deficiency negatively influenced de novo choline synthesis [29, 30, 37, 44].

The depletion of choline in the liver (both age groups) and kidney (adult group) tissues under folate deprivation observed in the present study can also be attributed to choline oxidation to betaine by choline dehydrogenase (EC 1.1.99.1) [14, 15, 37, 43]. Earlier studies reported high activity of this enzyme in the liver and kidney [45], whereas only negligible activity was observed in the brain [46, 47]. Furthermore, recent studies identified a distinct choline pool in liver and kidney tissue in mice that can be utilized under folate deprivation in combination with gamma radiation [48].

Choline is a precursor of acetylcholine [3, 11, 49]. Our results demonstrate a significant association between low choline levels in the heart and increased acetylcholine levels in response to folate deprivation in adult rats. Recent studies in rats suggest a strong association between increased acetylcholine levels in the heart and the development of hypertension [50, 51]. It was suggested that the increases in acetylcholine synthesis in the heart reflect augmented parasympathetic activity counteracting or compensating for the augmented sympathetic drive of early-stage hypertension [50, 51]. Our present data demonstrate that folate deficiency potentiates age-related increases in acetylcholine levels in the hearts of adult rats. Studies in rats [9, 10, 31, 32, 52, 53] and humans [54–57] show that folate deficiency results in hyperhomocysteinemia, which, in turn, is associated with the risk of hypertension.

The present study also found a significant association between decreases in choline levels in peripheral tissues and increases in plasma choline concentrations in adult rats fed a folate deficient diet, suggesting potential choline redistribution between tissues. These findings support recent studies in mice that demonstrate that choline is recycled in the liver and redistributed from kidney, lung, and intestine to liver and brain when the choline supply is attenuated [58]. Similar choline redistribution was observed in mice exposed to folate deficiency and gamma irradiation [48]. Because choline readily crosses the blood-brain barrier through an unsaturated facilitated-diffusion system, changes in plasma choline can produce parallel changes in brain choline and enhance the formation and release of acetylcholine [12, 17, 23, 27]. A supply of choline for the synthesis of acetylcholine is essential for the normal functioning of cholinergic neurons [17, 21, 59]. In the initial step of acetylcholine biosynthesis, choline is taken up from the extracellular space by a sodium-dependent high affinity uptake system (SDHACU) located predominantly in the terminals of cholinergic neurons [60]. Transfer of choline by SDHACU, into cholinergic terminals can be the rate-limiting step for the synthesis of acetylcholine [12, 17]. Recent studies show that sodium-dependent choline transport is correlated with the release and on-demand synthesis of acetylcholine, which reflects the status of cholinergic neuronal activity [12, 17]. Our results show that the absence of change in plasma choline levels in young rats fed a folate deficient diet was associated with non-significant changes in choline and acetylcholine levels in evaluated brain regions as compared to levels in the respective control group. Conversely, high plasma choline levels in adult rats fed a folate deficient diet were associated with differential changes in choline and acetylcholine content in different brain regions. Specifically, there were significant increases in choline and acetylcholine levels in the cortex and striatum under folate deprivation, whereas hippocampal acetylcholine was significantly depleted. No significant changes in hippocampal choline concentrations were observed in the adult rats under folate deprivation.

Earlier studies failed to find significant changes in the enzymes that regulate acetylcholine metabolism, including choline acetyltransferase (the enzyme for acetylcholine synthesis) and acetylcholinesterase (the acetylcholine-hydrolyzing enzyme), in several strains of rats fed folate deficient or folate-supplemented diets for 6 months [61]. Therefore, our findings of differential region-specific effects of folate deficiency on acetylcholine metabolism in adult rats can potentially be attributed to differential changes in the acetylcholine precursor (i.e., choline) in these brain regions.

One of the important findings of the present study is the differential effect of folate deprivation on dopamine release in the striatum with age. Our previous studies showed a significant age-related decline in striatal dopamine release [45, 62, 63]. The present data confirm our previous findings and show significantly lower levels of dopamine release in the striatum in the adult rats as compared to levels in young animals fed a control diet. Young rats fed a folate deficient diet did not show decreases in dopamine release, whereas in adult rats there was a dramatic increase in dopamine release. The increase in dopamine release in the striatum in adult rats was also associated with higher levels of acetylcholine. Previous studies have demonstrated that the striatum is densely innervated by cholinergic interneurons [64–67], suggesting a potential relationship between striatal acetylcholine and dopamine release [42, 67]. However, the mechanisms of this relationship remain largely unknown. Because one can assume that proper striatal functioning depends upon the precise interplay between the acetylcholine and dopamine systems, any age- or folate deficiency-related changes in this balance could subsequently involve a myriad of other neurotransmitters and neuromodulators and be translated ultimately into modification of cognitive functions.

Our results confirm the hypothesis that a folate deficient diet (FD) has a greater effect on the cholinergic system in the peripheral nervous system than in the brain, and that this effect escalates with age. To the best of our knowledge, this is the first comprehensive study to establish that both age groups have higher choline and acetylcholine metabolic sensitivities to short-term nutritional folate deficiency in the peripheral nervous system than they do in the brain. Furthermore, this study demonstrates that adaptation of choline and acetylcholine metabolism to folate deficiency in young rats appeared to be more efficient than in adult rats. These findings implicate adult rats as a preferred model for further investigations of molecular mechanisms of folate-choline-acetylcholine relationships.

A limitation of our study is its inability to determine whether changes in choline and acetylcholine metabolism in brain regions controlling behavior result in cognitive modifications in adult folate deficient rats. The rat number per experimental group was sufficient to observe significant changes in the cholinergic system. However, this number was significantly underpowered for the assessment of cognitive functions, the determination of which requires ten rats per group. Therefore, in a subsequent study we will use a larger number of adult rats per group and conduct cognitive assessments. Furthermore, cell culture studies must be performed in the future to provide underlying mechanistic data for some of the changes observed in the present whole animal approach.

Acknowledgment

The authors gratefully acknowledge Aniket Gurav and Mary Nashed for technical assistance and Language Editing Services listed on the Elsevier resource center website for editorial assistance. This research was supported by NIH grant AG009525 (JKB) as well as by ARS Cooperative Agreement with Tufts University #58-1950-9-935, CRIS # 1950-51000-070-10S.

Abbreviations

JM USDA HNRCA

Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging

FD

folate deficient diet

TBHQ

tert-Butylhydroquinone

HPLC

high-performance liquid chromatography

BRM

Krebs-Ringer basal release medium

ALT

alanine aminotransferase

AST

aspartate aminotransferase

SDHACU

sodium-dependent high affinity choline uptake system

Footnotes

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Disclaimer Statement

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. No authors report any actual or potential conflicts of interest.

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