Insulin-like growth factor I and its binding protein-3 are regulators of lactation and maternal responsiveness.

Adaptation to motherhood includes maternal behaviour and lactation during the postpartum period. The major organizing centres of maternal behaviour and lactation are located in the hypothalamic medial preoptic area (MPOA) and the arcuate nucleus, respectively. Insulin-like growth factor I (IGF-I) is an effector of the growth hormone axis; however, its function in the brain is largely unexplored. We identified increased maternal IGF binding protein-3 (IGFBP-3) expression in preoptic rat microarray data and confirmed it by RT-PCR. In situ hybridization histochemistry showed markedly elevated IGFBP-3 expression in the MPOA and the arcuate nucleus in rat dams. Prolonged intracerebroventricular injection of IGF-I or antagonism of brain IGFBP-3 with an inhibitor (NBI-31772) using osmotic minipumps increased pup retrieval time, suggesting reduced maternal motivation. Suckling-induced prolactin release and pup weight gain were also suppressed by IGF-I, suggesting reduced lactation. In addition, IGF-I-induced tyrosine hydroxylase expression and its specific phosphorylation in tuberoinfundibular dopaminergic neurons suppress prolactin secretion. Thus, IGF-I may inhibit both behavioural and lactational alterations in mothers. Neurons in the MPOA and arcuate nuclei express IGFBP-3 during the postpartum period to neutralize IGF-I effects. IGFBP-3 can prevent the blockade of maternal behaviour and lactation exerted by IGF-I, suggesting a novel modulatory mechanism underlying the behavioural and hormonal effects during central maternal adaptations.

Synaptic proteome changes in the hypothalamus of mother rats.

To establish synaptic proteome changes associated with motherhood, we isolated synaptosome fractions from the hypothalamus of mother rats and non-maternal control females at the 11th postpartum day. Proteomic analysis by two-dimensional differential gel electrophoresis combined with mass spectrometric protein identification established 26 significant proteins, 7 increasing and 19 decreasing protein levels in the dams. The altered proteins are mainly involved in energy homeostasis, protein folding, and metabolic processes suggesting the involvement of these cellular processes in maternal adaptations. The decrease in a significantly altered protein, complement component 1q subcomponent-binding protein (C1qbp) was validated with Western blotting. Furthermore, immunohistochemistry showed its presence in hypothalamic fibers and terminals in agreement with its presence in synaptosomes. We also found the expression of C1qbp in different hypothalamic nuclei including the preoptic area and the paraventricular hypothalamic nucleus at the protein and at the mRNA level using immunohistochemistry and in situ hybridization histochemistry, respectively. Bioinformatical network analysis revealed that cytokines, growth factors, and protein kinases are common regulators, which indicates a complex regulation of the proteome change in mothers. The results suggest that maternal responsiveness is associated with synaptic proteins level changes in the hypothalamus, and that growth factors and cytokines may govern these alterations. BIOLOGICAL SIGNIFICANCE: The period of motherhood is accompanied with several behavioral, neuroendocrine, emotional and metabolic adaptations in the brain. Although it is established that various hypothalamic networks participate in the maternal adaptations of the rodent brain, our knowledge on the molecular background of these alterations remains seriously limited. In the present study, we first determined that the functional alterations of the maternal brain can be detected at the level of the synaptic proteome in the hypothalamus. Independent confirmation of synaptic localization, and also the established decrease in the level of C1qbp protein suggest the validity of the data. Common regulators of altered proteins belonging to the growth factor and cytokine family suggest that the synaptic adaptation is governed by these extracellular signals and future studies should focus on their specific roles. Our study was also the first to describe the expression pattern of C1qbp in the hypothalamus, a protein potentially involved in mitochondrial and neuroimmunological regulations of synaptic plasticity. Its presence in the preoptic area responsible for maternal behaviors and also in the paraventricular hypothalamic and arcuate nuclei regulating hormonal levels suggests that the same proteins may be involved in different aspects of maternal adaptations. The conclusions of the present work contribute to establishing the molecular alterations that determine different maternal adaptations in the brain. Since maternal changes are models of neuronal plasticity in all social interactions, the reported results can affect a wide field of molecular and behavioral neuroscience.

Maternal alterations in the proteome of the medial prefrontal cortex in rat.

Proteomic differences between rat dams and control mothers deprived of their pups immediately after delivery were investigated in the medial prefrontal cortex (mPFC). A 2-D DIGE minimal dye technique combined with LC-MS/MS identified 32 different proteins that showed significant changes in expression in the mPFC, of which, 25 were upregulated and 7 were downregulated in dams. The identity of one significantly increased protein, the small heat-shock protein alpha-crystallin B chain (Cryab), was confirmed via Western blot analysis. Alpha-crystallin B chain was distributed in scattered cells in the mPFC, as demonstrated by immunohistochemistry. Furthermore, it was found to be localized in parvalbumin-containing neurons using double labeling. The elevation of its mRNA level in rat dams was also demonstrated via RT-PCR. The functional classification of the altered proteins was conducted using the UniProt and Gene Ontology protein databases. The identified proteins predominantly participate in synaptic transport and plasticity, neuron development, oxidative stress and apoptosis, and cytoskeleton organization. A common regulator and target analysis of these proteins determined using the Elsevier Pathway Studio Platform suggests that protein level changes associated with pup nursing are driven by growth factors and cytokines, while the MAP kinase pathway was identified as a common target. A high proportion of the proteins that were found to be altered in the mPFC are associated with depression. BIOLOGICAL SIGNIFICANCE: The behavior and emotional state of females change robustly when they become mothers. The brain, which governs these changes, may also undergo molecular alterations in mothers. As no proteomics approaches have been applied regarding maternal changes in the brain, we addressed this issue in the mPFC as this brain area is the uppermost cortical center of maternal control and the associated mood changes. The high number of protein-level alterations found between mothers taking care of their litter and those without pups indicates that pup nursing is associated with cortical protein-level changes. Alterations in proteins participating in synaptic transport, plasticity and neuron development suggest neuroplastic changes in the maternal brain. In turn, the relatively high number of altered proteins in the mPFC associated with depression suggests that the physiological effects of the protein-level alterations in the maternal mPFC could promote the incidence of postpartum depression. Cryab, a protein confirmed to be increased during maternal behaviors, was selectively found in parvalbumin cells, which, as fast-spiking interneurons, are associated with depression. The function of Cryab should be further investigated to establish whether it can be used to identify drug targets for future drug development.

Amylin is a novel neuropeptide with potential maternal functions in the rat.

Amylin, a 37-aa pancreatic peptide, was found to be expressed in the preoptic area of mother rats in our recent microarray study. Here, we report a marked increase in amylin expression around parturition and show that amylin mRNA level remains elevated as long as the pups are not removed from the dams. Amylin expression is also induced in maternally behaving (sensitized) nonlactating but not in nonsensitized nulliparous females or in females that did not become maternal despite the sensitization procedure. Immunohistochemistry verified the increased amylin peptide expression in maternally behaving rats and demonstrated the same expression pattern of amylin as in situ hybridization histochemistry. Ovariectomy had no effect on the activation of amylin neurons, suggesting sexual steroid-independent mechanisms. In subsequent functional experiments, mothers were separated from their pups for 22 h. On return of the pups, neuronal activation was found in the mother's preoptic area, with a distribution pattern similar to amylin-expressing neurons. Subsequent double labeling revealed that 86-93% of amylin neurons were activated by pup exposure. The results implicate amylin in the control of maternal adaptations, possibly exerting its actions on maternal behaviors via amylin receptors present in brain regions to which preoptic neurons project.