Comparison of the temporal programs regulating tyrosine hydroxylase and enkephalin expressions in TIDA neurons of lactating rats following pup removal and then pup return

Abstract

Dopamine (DA) and enkephalin (ENK) release from the tuberoinfundibular dopaminergic neurons (TIDA) into the hypophysial portal circulation is fundamentally different under non-lactating and lactating conditions. The aim of this experiment was to compare the effect of a brief interruption then resumption of suckling on the temporal program of tyrosine hydroxylase (TH; rate-limiting enzyme of dopamine synthesis) and ENK regulation in dams. On post partum day 10, pups were removed for a 4-h period from a group of the dams then returned for 4- and 24-h periods. It was examined whether such a brief interruption of suckling provokes full up-regulation of TH and down-regulation of ENK, and whether reinitiation of suckling limits the extent to which TH up- and ENK down-regulate. At the end of experiment, the animals were decapitated. In situ hybridization was used to examine the expression of TH and ENK mRNA in the arcuate nucleus where TIDA neurons reside. The results showed that, on one hand, the removal of pups induced TH up-regulation, on the other hand, ENK expression also increased 8 h after removal of pups and then started to slowly decline. In dams whose sucklings were reinitiated both TH and ENK mRNAs were up-regulated at least for a day. ENK expression responded more slowly to the removal of pups than expression of TH, and after reinitiation of suckling, the temporal program of regulation of both TH and ENK expressions ran parallel in the first 24 h.

Fig. 1

The graph chart shows optical density (OD) of TH mRNA clusters in TIDA neurons of A12 region of ARC in various experimental groups. Values are expressed as means±SE for each experimental group. The white bar represents the control group, in which the dams continued to lactate throughout the experiment without their pups being removed at all. The ‘no-return’ bars demonstrate the levels of OD at different times after pups were removed permanently. The ‘return’ bars demonstrate the OD levels at certain time points after pups were removed and then returned after a 4 h separation and continued suckling afterwards. The numbers in the bars indicate the number of rats used in each group. * indicates significant difference between continuously lactating group and the given ‘no-return’ group (p<0.05). # indicates significant difference between ‘return’ group and the corresponding ‘no-return’ group (p<0.05). α indicates significant difference between continuously lactating and the given return group (p<0.05)

Fig. 2

The graph chart shows the number of TH mRNA expressing cells detected in the A12 region of the ARC in the various experimental groups. Values are expressed as means±for each experimental group. The white bar is a control group, in which the dams continued to lactate throughout the experiment without their pups being removed at all. The ‘no-return’ bars demonstrate the mean of the number of cells of each group at various times after pups were removed permanently. The ‘return’ bars demonstrate the means of the number of cells at certain time points after pups were removed; however, these pups were returned after a 4 h separation and continued suckling afterwards. The numbers in the bars indicate the number of rats included in the group. * indicates significant difference between continuously lactating group and all the other groups (p<0.05)

Fig. 3

The graph chart demonstrates the optical density (OD) of ENK mRNA clusters in the A12 region of ARC in various experimental groups. Values are expressed as means±SE for each experimental group. The ‘no-return’ bars demonstrate the levels of OD at the indicated times after pups were removed permanently. The ‘return’ bars indicate the levels of OD at time points after pups were removed, but returned after a 4 h separation. The white bar indicates the OD of ENK mRNA in dams that continued to lactate and remained with their pups throughout the experiment. The numbers in the bars indicates the number of rats included in the group. * indicates significant difference between continuously lactating group and the given ‘no-return’ group (p<0.05). # indicates significant difference between ‘no-return’ group (7–8 h) and ‘no-return’ groups (48 and 72 h; p<0.05). α indicates significant difference between continuously lactating and the given return group (p<0.05)

Fig. 4

The graph chart shows the number of ENK mRNA expressing cells detected in the A12 region of the ARC in various experimental groups. Values are expressed as means±SE for each experimental group. The white bar is a control group, in which the dams continued to lactate throughout the experiment without their pups being removed at all. The ‘no-return’ bars demonstrate the mean of the number of cells of each group at certain times after pups were removed permanently. The ‘return’ bars demonstrate the means of the number of cells at certain time points after pups were removed; however, these pups were returned after a 4 h separation and continued suckling afterwards. The numbers in the bars indicate the number of rats included in the group. * indicates significant difference between continuously lactating group and the given ‘no-return’ group (p<0.05)

Fig. 5

Microphotographs demonstrating TH and ENK mRNA clusters in a continuously lactating dam (a and c) and in dams whose pups were removed (b and d). Arrows show mRNA clusters in TH and ENK expressing cells. The number and intensity of silver grains indicating TH and ENK expressions are markedly higher in dams whose pups were removed than in the continuously suckling dams. 3V third ventricle; ARC arcuate nucleus. Scale 50 μm

Fig. 6

Dynamics of changes in TH and ENK mRNA optical densities (OD) in ARC during lactation upon removal of pups (a) and upon removal and then return of pups (b)