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. 2017 Jan 10;10:240.
doi: 10.3389/fnbeh.2016.00240. eCollection 2016.

Dietary Prebiotics and Bioactive Milk Fractions Improve NREM Sleep, Enhance REM Sleep Rebound and Attenuate the Stress-Induced Decrease in Diurnal Temperature and Gut Microbial Alpha Diversity

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Dietary Prebiotics and Bioactive Milk Fractions Improve NREM Sleep, Enhance REM Sleep Rebound and Attenuate the Stress-Induced Decrease in Diurnal Temperature and Gut Microbial Alpha Diversity

Robert S Thompson et al. Front Behav Neurosci. .
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Abstract

Severe, repeated or chronic stress produces negative health outcomes including disruptions of the sleep/wake cycle and gut microbial dysbiosis. Diets rich in prebiotics and glycoproteins impact the gut microbiota and may increase gut microbial species that reduce the impact of stress. This experiment tested the hypothesis that consumption of dietary prebiotics, lactoferrin (Lf) and milk fat globule membrane (MFGM) will reduce the negative physiological impacts of stress. Male F344 rats, postnatal day (PND) 24, received a diet with prebiotics, Lf and MFGM (test) or a calorically matched control diet. Fecal samples were collected on PND 35/70/91 for 16S rRNA sequencing to examine microbial composition and, in a subset of rats; Lactobacillus rhamnosus was measured using selective culture. On PND 59, biotelemetry devices were implanted to record sleep/wake electroencephalographic (EEG). Rats were exposed to an acute stressor (100, 1.5 mA, tail shocks) on PND 87 and recordings continued until PND 94. Test diet, compared to control diet, increased fecal Lactobacillus rhamnosus colony forming units (CFU), facilitated non-rapid eye movement (NREM) sleep consolidation (PND 71/72) and enhanced rapid eye movement (REM) sleep rebound after stressor exposure (PND 87). Rats fed control diet had stress-induced reductions in alpha diversity and diurnal amplitude of temperature, which were attenuated by the test diet (PND 91). Stepwise multiple regression analysis revealed a significant linear relationship between early-life Deferribacteres (PND 35) and longer NREM sleep episodes (PND 71/72). A diet containing prebiotics, Lf and MFGM enhanced sleep quality, which was related to changes in gut bacteria and modulated the impact of stress on sleep, diurnal rhythms and the gut microbiota.

Keywords: MFGM; lactoferrin; nutrition; prebiotics; sleep; stress; temperature.

Figures

Figure 1
Figure 1
Timeline. Schematic depicting the experimental timeline. Rats arrived on postnatal day (PND) 24 and were immediately placed on either the control or test diets. Fecal samples for 16S rRNA analysis were collected at PND 35, PND 70 and PND 91. Biotelemetry devices were implanted at PND 59 and 10 days later sleep, body temperature and locomotor activity (LA) measures were continuously recorded until the end of the experiment. On PND 87 rats were exposed to inescapable stress and were placed back in home cages to monitor how the test diet modulated sleep recovery following stress exposure. Finally, body weights, food weights and fecal samples were taken on PND 91 to assess the impact of stress exposure on gross physiology measures and the gut microbiota structure.
Figure 2
Figure 2
Test diet increases Lactobacillus rhamnosus. Data demonstrating, in a subset of rats, that consumption of the test diet significantly increased levels of Lactobacillus rhamnosus in fecal cultures at PND 52, when compared with those eating the control diet (#p < 0.05 vs. control diet).
Figure 3
Figure 3
Stress effects on food/body weight/diurnal CBT. Food consumption, body weight and diurnal amplitude data 4 days after stress exposure. (A) Stress exposure reduced food consumption, but there were no differences in food consumption between the control and test diets. The food consumption depicted was a weekly average based on food consumed from PND 85–91. (B) Similarly, stress exposure resulted in lower body weights but there were no differences in body weight between the control and test diets. (C) Stress flattened the diurnal amplitude of LA of both control and test diet groups, but diet did not alter this effect. (D) Stress also flattened the diurnal amplitude of CBT of both control and test diet groups, however, the test diet group had an attenuated disruption in the diurnal amplitude of CBT when compared with those on the control diet. Abbreviations are as follows: a.u., arbitrary units; CBT, core body temperature; g, grams; LA, locomotor activity; PND, post natal day (*p < 0.05 vs. no stress; #p < 0.05 vs. control diet).
Figure 4
Figure 4
Diet effects on sleep. Data expressed as average amounts of the two recording days (12 h light and 12 h dark) indicating that rats consuming the test diet spent more time in NREM sleep and had increased NREM sleep consolidation in early-life. (A) Wake was reduced and (B) NREM sleep was increased during the light cycle in rats consuming test diet when compared to rats on the control diet. (C) There was no effect of diet on rapid eye movement (REM) sleep. (D) The average duration of NREM sleep episodes was significantly longer indicating greater NREM sleep consolidation in rats consuming the test diet when compared to rats on the control diet. One week later (E) wake was still reduced and (F) NREM sleep increased during the light cycle in rats consuming test diet, (G) there was no effect of diet on REM sleep, and (H) there was only a trend (p = 0.07) towards increased NREM sleep consolidation. The effects of diet were no longer present on (I) wake, (J) NREM sleep, (K) REM sleep or (L) NREM episode duration on PNDs 85, 86. Abbreviations are as follows: NREM, Non-rapid eye movement; PND, post natal days; sec, seconds (#p < 0.05 vs. control diet).
Figure 5
Figure 5
Day of stress. Data depicting that stress significantly disrupted sleep and physiology and that test diet enhanced REM sleep rebound following stress exposure. (A) REM sleep was abolished during stress and increased during the ensuing dark cycle. Rats consuming the test diet displayed enhanced REM rebound (yellow shading for clarity) when compared to stress rats eating the control diet. (B) NREM sleep was also abolished during stress and was increased compared to non-stressed rats in the ensuing dark cycle. (C) Wake was at a maximum during stress exposure and stressed rats spent less time awake during the ensuing dark cycle (due to increased sleep). (D) Rats exposed to stress also were less active during the nocturnal cycle following stress. (E) Stress induced a hyperthermic response in rats, which persisted for several hours following stress during the remaining light cycle, but was reversed to below the non-stressed controls upon the dark cycle onset (likely due to increased sleep in the stressed rats). Abbreviations are as follows: a.u., arbitrary units; NREM, Non-rapid eye movement; REM, Rapid eye movement. Note: shaded area of graph is for clarification and does not represent “actual” time stressed (see “Materials and Methods” Section) (*p < 0.05 vs. no stress; #p < 0.05 vs. control diet).
Figure 6
Figure 6
16S rRNA data. Taxonomic graphs depicting the relative abundance of phyla in the gut microbial communities at PND 35, PND 70 and PND 91. (A) The high abundance gut microbial community at PND 35 in rats consuming either the control or test diet. (B) The low abundance microbial communities depicted in the absence of bacteroidetes, firmicutes and proteobacteria. (C) The high abundance microbial community at PND 70 in rats consuming either the control or test diet. (D) The low abundance microbial communities depicted in the absence of bacteroidetes, firmicutes and proteobacteria. (E) The high abundance gut microbial community at PND 91 4 days after stress exposure in rats consuming either the control or test diet. There was a small, but significant, increase in the phylum bacteroidetes (orange bars). (F) The low abundance microbial communities depicted in the absence of bacteroidetes, firmicutes and proteobacteria 4 days after stress exposure. Abbreviations are as follows: OTUs, operational taxonomic units; PND, post natal days (*p < 0.05 vs. no stress groups).
Figure 7
Figure 7
Alpha diversity. Data depicting three measures of alpha diversity. (A) Stress exposure reduced the number of observed species in rats on the control diet, but rats on the test diet were protected from this reduction in the number of observed species. (B) Stress exposure reduced Shannon Entropy in rats on the control diet, but this effect was attenuated in rats consuming the test diet. (C) Similarly, stress reduced PD whole tree analysis, but only in rats on the control diet. Abbreviations are as follows: PD, phylogenetic diversity; PND, post natal days (*p < 0.05 vs. control diet, no stress).
Figure 8
Figure 8
Multiple regressions. The relationship between early-life gut microbiota and sleep was examined by multiple regression analyses. (A) Prior to stress exposure, in the control diet group there was no significant relationship between Deferribacteres at (PND 35) and NREM episode duration at PND 71, 72 denoted by the dotted line. In the test diet group, however, there was a significant linear relationship between Deferribacteres and NREM episode duration such that rats consuming the test diet with lower levels of Deferribacteres (PND 35) had longer NREM episode durations denoted by the solid black line (p = 0.016). (B) Following stress exposure there was a small, but significant, linear relationship between Proteobacteria at PND 35 and REM recovery sleep at PND 87 denoted by the solid black line (p = 0.030). Abbreviations are as follows: PND, post natal days.
Figure 9
Figure 9
Phylum Deferribacteres. Rats consuming the test diet had significantly lower levels of Deferribacteres at PND 35 when compared to rats consuming the control diet. Abbreviations are as follows: OTUs, operational taxonomic units; PND, post natal days (#p < 0.05).

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