An autoradiographic analysis of rat brain nicotinic receptor plasticity following dietary choline modification

Abstract

Choline is known to be involved with numerous physiological functions of the nervous system and also acts as a direct acting agonist of alpha7 nicotinic acetylcholine receptors (nAChRs). The purpose of this study was to conduct a brain region-specific evaluation of changes in nAChR subtype expression following dietary choline modification. In addition, we assessed changes in body weight, food/water intake, as well as changes in spatial learning (Morris Water Maze) in response to dietary choline modification. Male Sprague-Dawley rats were exposed to standard, choline supplemented or choline deficient diets for periods of 14 or 28 days. Choline supplemented animals gained significantly less weight over the course of the experiment, in spite of the fact that there were minimal differences in food consumption between the dietary regimens. Spatial memory did not differ between animals maintained on a standard rat diet, and the choline supplemented food. Brains of the animals kept on the diets for 14 and 28 days were used for quantitative autoradiographic analysis of nicotinic receptor subtypes using 125I-Bungarotoxin (alpha7) and 125I-Epibatidine (non-alpha7). There were no significant differences in nicotinic receptor binding or physiologic parameters measured between animals fed standard and choline deficient diets. However 2 weeks of dietary choline supplementation caused significant up-regulation of alpha7 receptors without significant effect on the density of non-alpha7 nAChRs. Increases in BTX binding predominantly occurred in cortical and hippocampal brain regions and ranged between 14 and 30% depending on the brain region. The results of our study suggest that choline acts as a selective agonist at alpha7 nicotinic cholinergic receptors in the rat central nervous system.

Figure 1

Average daily food intake in animals exposed to diets with varying choline content for 28 days. Repeated measures ANOVA showed significant effects of diet (F=3.42; P=0.049) and time (F=84.0; P<0.0001). Post-hoc Tukey procedure demonstrated a significant difference in food intake between animals consuming a choline-supplemented diet compared to the standard choline diet group on Day 2 of the experiment (* P<0.05). Significant differences in food consumption between choline supplemented and choline deficient groups were detected on days 2 and 4 of the study. Beyond day 4 of the experiment, there were no group differences in food consumption noted. Error bars are omitted from this graph to avoid obstruction of the data points.

Figure 2

Average water intake in animals exposed to diets with varying choline content for 28 days. ANOVA results showed significant effects of diet (F=12.2; P=0.0002) and time (F=81.8; P<0.0001) on daily water consumption. While there were no group differences in fluid consumption over the first 2 weeks of the study, Post-hoc Tukey procedure demonstrated that choline supplemented animals had significantly increased water consumption on most days in the last 2 weeks of the study (* P<0.05 vs standard choline diet; † P<0.05 vs choline deficient diet). Error bars are omitted from this graph to avoid obstruction of the data points.

Figure 3

Average body weight gain, assessed approximately every other day in animals exposed to diets with varying choline content for 28 days. ANOVA revealed significant increase in weight as time went on (F=51.62; P<0.0001) as well as a significant effect of dietary treatment (F=15.31; P<0.0001). Post-hoc analysis indicated that choline supplemented animals gained significantly less weight than animals consuming either the standard choline or choline deficient diets. Error bars are omitted to avoid obstruction of the data points.

Figure 4

Two weeks of dietary choline supplementation does not alter cognitive performance in the acquisition phase of Morris Water Maze testing. ANOVA demonstrated a significant effect of the training day (F=39.8, P<0.0001), but no significant effect of dietary regimen or diet × day interaction. A retention (probe) test was conducted four hours following the final acquisition trial, but there were no significant effects of dietary treatment (data not shown).

Figure 5

Four weeks of dietary choline supplementation does not alter cognitive performance in the acquisition phase of Morris Water Maze testing. ANOVA demonstrated a significant effect of the training day (F=39.8, P<0.0001), but no significant effect of dietary regimen or diet × day interaction. Animals fed a choline deficient diet and animals consuming a choline-supplemented diet performed similar to animals consuming a standard choline diet on all test days. A retention (probe) test was conducted four hours following the final acquisition trial, but there were no significant effects of dietary treatment (data not shown).

Figure 6

Representative autoradiographs depicting α-[125I]-BTX binding in animals exposed to a standard choline diet (left column), a choline deficient diet (middle column) or a choline supplemented diet (right column) for 14 days. Choline supplemented animals exhibited significantly higher BTX binding in some brain regions compared to animals that consumed the standard choline or choline deficient diets. Cx 1–4, cerebral cortex layers 1–4; Cx 5–6, cerebral cortex layers 5–6; DG, dentate gyrus; En, endopiriform nucleus; Hi, hilus of the dentate gyrus; PH, posterior hypothalamus SC, superior colliculus; SO, stratum oriens; STh, subthalamic nucleus; VLG, ventrolateral geniculate nucleus.