Cocaine- and amphetamine-regulated transcript (CART) expression is differentially regulated in the hypothalamic paraventricular nucleus of lactating rats exposed to suckling or cold stimulation

Abstract

Neural stimuli, such as suckling or cold exposure, increase TRH mRNA in the paraventricular nucleus (PVN) of the rat hypothalamus, yet only suckling induces prolactin secretion. As TRH co-localizes with cocaine- and amphetamine-regulated transcript (CART) in hypophysiotropic neurons of the PVN, and CART inhibits TRH-induced prolactin release but not TRH-induced TSH release in adenohypophyseal cell cultures, we raised the possibility that differential regulation of CART gene expression in the PVN may explain the differences in prolactin secretion following each of the two stimuli. Primiparous female rats were mated and handled daily during the pre- and postpartum periods. After delivery, the litter was adjusted to 8 pups and at mid-lactation, dams were separated from their pups for 8 h and exposed to either 1 h of cold or 30 min of suckling. Long-term effects of suckling were studied by separating pups from their mothers for 24 h, followed by a 12 h period of continuous suckling. Serum TSH levels increased in response to cold exposure, while prolactin levels were increased by suckling and diminished by cold exposure. CART mRNA levels increased in rostral and mid parts of the medial parvocellular PVN following cold exposure but not after suckling stimulation. These data demonstrate a differential regulation of CART gene expression in hypophysiotropic neurons in response to stimuli that increase TRH mRNA levels, and suggest that CART activation in the PVN may contribute to the decrease in PRL release when the thyroid axis is activated by cold exposure.

Figure 1

Serum hormone concentrations of control, suckling and cold stimulated lactating females quantified by radioimmunoassay (5 animals per group). Values represent mean ± sem. (* P < 0.05 or ** P < 0.001 statistically different by Newman-Keuls post hoc testing)

Figure 2

Semiquantitative RT-PCR for CART mRNA and TRH mRNA in the PVN of lactating rats exposed to cold (A, C) or suckling (B, D) stimulation. Densitometric analysis showed a significant increase in the PVN for CART mRNA after 1hr of cold stimulation and a tendency to decrease after 30’ of suckling (optical density = Arbitrary units). TRH mRNA was significantly increased after cold or suckling stimulation. A total of 5 animals per group were analyzed in each condition. t-Test * P < 0.05 statistically different from control RT animals

Figure 3

Distribution of CART mRNA in the parvocellular PVN of lactating rats exposed to 30 min of suckling (column C) or 1h of cold exposure at 4C (column D). CART mRNA was detected by in situ hybridization using a ³⁵S -labeled probe. Note the increase in the density of silver grains in cold exposed animals in the rostral (1st row) and mid (2^nd row) sections of the parvocellular PVN (paPVN). First column (A): bright-field illumination showing representative hematoxilin-eosin stained sections trough the rostro-caudal paPVN. Second column (B): CART mRNA pattern in control RT lactating rats; third (C), in lactating rats suckled for 30 min (S30) and last (D) on lactating rats exposed 1h to cold, without pups. Graphs show semiquantitative analysis of CART mRNA after 30’ of suckling or 1hr of cold exposure. Values represent mean ± sem from 4, 8 or 4 slices/animal/group in the rostral, mid and caudal paPVN respectively. (* P < 0.05 or ** P < 0.01 statistically different from control RT animals; Newman-Keuls post hoc testing)

Figure 4

TRH and CART mRNA expression in the PVN of lactating females separated from their pups 24 h on day 12, and then reunited with them for a 12 h period. TRH and CART mRNA were detected by in situ hybridization using ³⁵S-labeled probes. Note the significant increase in silver grains for TRH mRNA after 12 h of continuous suckling in the mid PVN while no changes can be detected for CART mRNA.