Nature's food anticipatory experiment: entrainment of locomotor behavior, suprachiasmatic and dorsomedial hypothalamic nuclei by suckling in rabbit pups

Abstract

In nature and under laboratory conditions, dams nurse rabbit pups once daily for a duration of fewer than 5 min. The present study explored neural mechanisms mediating the timing of nursing in this natural model of food anticipatory activity, focussing on the suprachiasmatic nucleus (SCN), the locus of the master circadian clock and on the dorsomedial hypothalamic nucleus (DMH), a region implicated in timing of food-entrained behavior. Rabbit pups are born in the dark, with eyelids closed. Nursing visits to the litters also occurs during the dark phase. To explore the effect of the timing of feeding, pups were maintained in constant darkness, while females housed in a light-dark cycle were permitted to nurse their pups either during the night (night-fed group) or day (day-fed group). All pups exhibited anticipatory locomotor activity before daily nursing. In the SCN, PER1 and FOS peaked during the night in both groups, with a longer duration of elevated protein expression in the night-fed group. In contrast, DMH peak PER1 expression occurred 8 h after pups were fed, corresponding to the shift in timing of nursing. Comparison of nursed and 48 h fasted pups indicates that the timing of PER1 expression was similar in the SCN and DMH, with fewer PER1-positive cells in the latter group. The results indicate that rabbit pups show food anticipatory activity, and that timing of nursing differentially affects PER1 expression in the SCN and DMH.

Fig. 1

Actograms show free running locomotor activity of male (left) and female (right) mice housed in constant darkness. The horizontal axis is 48 h and the vertical axis is consecutive days. Each box shows an individual animal before and after an experimental manipulation. The top two panels show intact, then GDX/OVX male and female mice. The bottom two panels show individual GDX/OVX animals that are then treated with either TP and DHT, respectively.

Fig. 2

The bar graphs show quantification of the effects of GDX/OVX and hormone replacement in male (left column) and female (right column) mice, as well as sex differences on several behavioral measures. Each of the measures (Period, Precision, Activity Duration and Amount of activity) is presented for each sex. For Amount of activity, histograms sharing common letters are not statistically different from each other. The presence of a black or white circle at the bottom of the bar indicates a measure in which the sexes differ significantly (p<0.05) from each other. *p<0.05, **p<0.01, ***p<0.001.

Fig. 3

Analysis of % of daily activity levels in each of four epochs, each lasting 6 h, in male and female (A) intact, (B) GDX/OVX, (C) GDX/OVX+TP, (D) GDX/OVX+DHT mice. The offset of activity was taken as circadian time 24 (see Methods for further explanation). ***p<0.001.

Fig. 4

Western blot analysis of AR protein levels in male and female SCN punches indicate that males have about two-fold more AR protein in the SCN than do females, ***p<0.001.

Fig. 5

(A) Photomicrographs show fluorescent double-labeled AR- (red) and AVP-ir (green) in SCN of intact male and female mice. Note that AR-ir is localized to the core SCN region lacking AVP-ir. Upper panel shows AVP, middle panel shows AR, and lower panel shows overlay. (B) Photomicrographs show AR-ir in GDX/OVX (upper panel) and GDX/OVX+TP (lower panel) treated male and female mice.

Fig. 6

Histograms show number of AR-ir cells (A) in intact males and estrus and diestrus females and (B) GDX male and OVX female and TP-treated mice. **p<0.01, ***p<0.001.