Mastication stimuli regulate the heartbeat rate through rhythmic regulation by the hypothalamic-autonomic system; molecular and telemetric studies in weaning-stage rats

Figures

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  Figure 1. Animal-raising schedule and apparatus. (A) Time course of the experiment. In experiment 1 (Exp. 1), 3 week-old male rats (n = 5) were acclimatized from days −2 to 0 with a powder diet, raised under different mastication conditions (powder diet and chow diet) from days 0 to 8, and sacrificed on day 8. In experiment 2 (Exp. 2), rats (n = 3) were implanted with transmitters on day −2, acclimatized from day −2 to 0, and the heart muscle potential was measured under the same conditions used in Exp. 1 from day 0 to 12. (B) Total food intake from days 0 to 20 (time of sacrifice). (C) Total water intake from day 0 to 22. (D) Total weight gain over 22 days (days 0 to 22).

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  Figure 2. Regulation of the hypothalamic transcriptome by mastication stimuli. (A) Five samples from each experimental group (Powder or Chow) were subjected to DNA microarray analysis and represented in a hierarchical cluster. (B) The volcano plot of the differentially expressed genes with the number of probe sets showing significant differences in the expression levels between the Powder and Chow groups (FDR < 0.05). Because the genes were extracted by the non-parametric statistics (rank products), some genes were plotted in the opposite quadrant. The FDR values less than 0.0001 were not provided by the program, therefore they were plotted at FDR = 0.00005.

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  Figure 3. Time course of animal activity and heartbeat rate. (A) In-day traces of animal activity within 5 min on day 8. Gaps indicate disturbances in data acquisition due to animal maintenance between hours 12 and 15 (12.15). (B,C) Average animal activity during the light or dark period from days 1 to 12. (D,E) Averages of the heartbeat rate. Inset tables; The results of repeated two-way ANOVA with significant p-values in boldface (p < 0.05). *Significant difference was detected using the t-test (p < 0.05).

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  Figure 4. Time course of low frequency (LF), high frequency (HF) band integration, and LF/HF ratio of the heart rate power spectrum. (A–F) Average light and dark period data from day 1 to 12. Inset tables; The results of repeated two-way ANOVA with significant p-values in boldface (p < 0.05). *Significant difference was detected using the t-test (p < 0.05).

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  Figure 5. Time course of the heartbeat rate fluctuation indexes. (A,B) Averages of the standard deviations of R wave-R wave intervals within 5 min (SDRR) from day 1 to 12. (C,D) Averages of the standard deviation of the root mean square successive difference of R wave-R wave intervals within 5 min (RMSSD). Inset tables; The results of repeated two-way ANOVA with significant p-values in boldface (p < 0.05). *Significant difference was detected using the t-test (p < 0.05). (E,F) Correlation between the SDRR and RMSSD values on day 8 of the light and dark periods. Each dot represents the values obtained every 5 min from six rats. The solid and dotted lines represent linear approximations of the C and P groups, respectively. The Pearson’s correlation coefficient (r) was calculated for each experimental group.

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  Figure 6. Regulation of the heartbeat rate by mastication stimuli during the weaning period. Intermittent mastication stimuli activate both total autonomic and parasympathetic activation through the hypothalamus, resulting in a decreased heart rate at 5 week-old. Avp, arginine vasopressin; Cart, cocaine-amphetamine-regulated transcript pre-propeptide; Crh, corticotropin-releasing hormone; Trh, thyrotropin-releasing hormone; Hcrt, hypocretin neuropeptide precursor.