Locomotor behavior
Rats under AL and AL-F conditions showed an increase of locomotor behavior at the beginning of the night which is the onset of their normal activity phase. In general, it was low during the day with a sharp increase at ZT13 at the onset of the night and remained high along the dark phase. RF and RF-F groups had a completely different pattern. Rats under restricted feeding, RF and in RF-F groups developed FAA at the middle of the day at the time when they are normally sleeping. As shown in the actogram (Figure 1a) the locomotor behavior started increasing about 2 h before the mealtime (ZT3-4), reaching the maximal activity at the moment of food access. In both groups the highest value at ZT5 (at mealtime for RF or projected mealtime RF-F), was significantly higher than the time point before (ZT1; p< .05) and after feeding (ZT7; p< 0.05). The two-way ANOVA indicated that locomotor activity varied significantly among feeding conditions (F3-48=11.64, P< .001), time factor (F3-48= 28.72, p< .001) and the interaction between feeding condition and time (F3-48=11.52, P< .001; (Fig. 1b). The post hoc analysis between groups indicated that values at ZT5 for RF and RF-F groups were significantly higher than AL and AL-F conditions (p< .001).
Supraoptic nucleus (SON)
In the SON food intake induced an increase of c-Fos and also an activation of OT cells after feeding in food-entrained rats.
In the AL group, OT values remained constant except at ZT5, which was significantly lower than value at ZT1 (p=0.02). In the RF group, a significant increase at ZT7 was observed in comparison to values before feeding at ZT1 and at ZT5 (P<0.01). In the RF-F group the increase at ZT7 did not persist; values were similar in all time points except at ZT7, which was significantly lower than values at ZT1 and at ZT13 (p<0.05). In the AL-F group, OT values were in general higher than in the other groups. The two-way ANOVA indicated that OT expression in the SON varied significantly due to the group condition (F3-63=37.98, P<0.001), and the interaction between feeding condition and time (F9-63 = 9,011, P<0.001), but not by time factor (F3-63 = 2,55 P<0.06; Fig. 2a).
c-Fos values were low in all groups except in RF group where food intake induced a sharp increase of this protein at ZT7 (P<0.001). This activation by food ingestion did not persist in the RF-F group, which exhibited similar values as the AL group. In the AL-F group, the c-Fos expression was also low and similar to the AL in all time points. The two-way ANOVA indicated that c-Fos expression in the SON varied significantly with group condition (F3-63 = 442.94; P<0.001), due to the time factor (F3-63 = 232.79, P<0.001) and to the interaction between feeding condition and time (F9-63 = 233.21, P<0.001; Figure 2b.
Double-labelled c-Fos/OT cells were scarce in all groups except in the RF group where food intake induced at ZT7 (p<0.001) a sharp increase of double-labelled cells (Figure 2c). In the RF-F group, the food entrained increase at ZT7 did not persist; the OT % activation was similar than in AL and AL-F groups. The two-way ANOVA indicated that the c-Fos/OT percentage of activation in the SON varied significantly among groups condition (F3-63 = 483.75, P<0.001), time factor (F3-63 = 221.37, p<0.001) and the interaction between groups and time (F9-63 = 193.79, p<0.001; Figure 2c). In Figure 3a and b we show microphotographs of SON at the time of feeding (ZT5) and two hours later at ZT7.
Median subregion of the Paraventricular hypothalamic nucleus (PVNm)
Similar as observed in the SON, in the PVNm food intake induced an increase of c-Fos and also an activation of OT cells after feeding in food-entrained rats.
In the AL group, the number of OT positive cells was similar in all time points with only a significant increase at ZT13 in comparison to ZT1 and at ZT5 values (p<0.001). In the RF group, there was an increase of OT expression at ZT7 that was significantly different from the remaining time points (P<0.001). In the RF-F group, values remained unchanged in most time points except at ZT13 which was significantly lower from remaining time points (p<0.001). In the AL-F group, OT value at ZT7 was significantly higher than remaining time points (p<0.001 in all cases). The two –way ANOVA indicated that OT expression in the PVNm varied significantly with group condition (F3-63 = 235.289, p<0,001), time factor (F3-63 = 56.755, p<0,001) and the interaction between feeding condition and time (F9-63 = 36.760, p<0,001; Figure 4a).
c-Fos values were low in all groups except in RF group where food intake induced a sharp increase of this protein at ZT7 (p<0.001; Figure 4b). This increase did not persist in the RF-F group, which had low values at all time points similar to AL and AL-F groups. The two-way ANOVA indicated that in the AL, RF, RF-F and AL-F groups, c-Fos expression in the PVNm varied significantly with group condition (F3-63 = 2847.694, p<0,001), time factor (F3-63 = 3153.575, p<0,001) and the interaction between feeding condition and time (F9-63 = 3263.682, p<0,001).
In the PVNm the AL group showed high c-Fos/OT expression at ZT1 significantly different from the remaining time point values (p<0.001; Figure 4c). In the RF group, food ingestion triggered expression of c-Fos in OT cells at ZT7 and ZT13, which were significantly higher than the values at ZT1 and ZT5 (p<0.001) and also from all groups at ZT7 and ZT13 (P<0.001). The increase in activation of OT cells did not persist in the RF-F group. In this group the highest OT % activation was observed at ZT1, which was significantly different from the remaining time point values (P<0.001 in all cases). In the AL-F group, the OT % activation was not significantly different at any time point (p=0.250). The two-way ANOVA indicated that OT % activation in the PVNm varied significantly with group condition (F3-63 = 335.60, P<0,001), time factor (F3-63 = 74.92, p<0,001) and the interaction between feeding condition and time (F9-63 = 116.54, p<0,001; Figure 4c). In Figure 3c and d we show microphotographs of the PVNm at the time of feeding at ZT5 and two hours after at ZT7.
Posterior subregion of the paraventricular hypothalamic nucleus (PVNp)
In contrast to the SON and PVNm, the PVNp exhibited a completely different pattern of c-Fos activation in OT cells. In the RF group, there was a sharp induction of c-Fos before food intake, at the time of FAA. Importantly, this increase persisted in the RF-F group in fasted conditions at the time of the previous scheduled mealtime.
The number of OT immunoreactive cells was constant along time points for all experimental groups (Figure 5a). The two-way ANOVA indicated that OT expression in the PVNp did not vary with group condition (F3-63 =1.48, p=0.23), time factor (F3-63 = 1.39, p=0.25) nor with the interaction between feeding condition and time (F9-63 =0.54, p=0.83).
The c-Fos expression in the AL group was not significantly different at any time point (p=0.146). In the RF group, a high c-Fos expression was observed at ZT7, significantly higher than the other time points (p<0.001). In the RF-F and AL-F groups, c-Fos did not significantly vary among time points (P=0.203 and p=0.753, respectively).
The two-way ANOVA indicated that c-Fos expression in the PVNp varied significantly with group condition (F3-63 = 9.83, P<0.001), time factor (F3-63 = 4.60, p=0.007) and the interaction between feeding condition and time (F9-63 = 9.94, P<0.001; Figure 5b).
In contrast to the SON and PVNm, the PVNp showed an activation of c-Fos/OT cells before feeding at ZT5 coinciding with the time of FAA, and this activation persisted in the RF-F group (P<0.01; Figure 5c). In addition, food ingestion also showed an increase in activation of OT cells at ZT7, which was significantly higher than the values at ZT1 and ZT13 (P<0.01). This effect was not observed in AL and in AL-F groups (Figure 5c).
Double-labelled c-Fos/OT cells in the PVNp at ZT5 are shown in Figure 6a and b and their persistence in RF-F in Figure 6c and 6d). No double-labelled c-fos/OT cells were observed at ZT5 in the AL-F group (Figs. 6e, f). The two-way ANOVA indicated that c-Fos/OT positive cells in the PVNp varied significantly with group condition (F3-64 = 24.60, p<0,001), time factor (F3-63 = 39,18, P<0.001) and the interaction between feeding condition and time (F9-63 = 21.78, P<0.001).