Endogenous CNS expression of neurotensin and neurotensin receptors is altered during the postpartum period in outbred mice.

Neurotensin (NT) is a neuropeptide identical in mice and humans that is produced and released in many CNS regions associated with maternal behavior. NT has been linked to aspects of maternal care and previous studies have indirectly suggested that endogenous NT signaling is altered in the postpartum period. In the present study, we directly examine whether NT and its receptors exhibit altered gene expression in maternal relative to virgin outbred mice using real time quantitative PCR (qPCR) across multiple brain regions. We also examine NT protein levels using anti-NT antibodies and immunohistochemistry in specific brain regions. In the medial preoptic area (MPOA), which is critical for maternal behaviors, mRNA of NT and NT receptor 3 (Sort1) were significantly up-regulated in postpartum mice compared to virgins. NT mRNA was also elevated in postpartum females in the bed nucleus of the stria terminalis dorsal. However, in the lateral septum, NT mRNA was down-regulated in postpartum females. In the paraventricular nucleus of the hypothalamus (PVN), Ntsr1 expression was down-regulated in postpartum females. Neurotensin receptor 2 (Ntsr2) expression was not altered in any brain region tested. In terms of protein expression, NT immunohistochemistry results indicated that NT labeling was elevated in the postpartum brain in the MPOA, lateral hypothalamus, and two subregions of PVN. Together, these findings indicate that endogenous changes occur in NT and its receptors across multiple brain regions, and these likely support the emergence of some maternal behaviors

Non-significant virgin and postpartum IHC mean density and one-way ANOVA results for each region or subregion tested.

<p>Notes: The sample numbers listed above were the final sample numbers used for data analysis. A complete list of original sample numbers for each brain region can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083098#s5" target="_blank">Methods</a> section. Missing or torn sections led to some regions having fewer samples than others.</p><p>^# indicates that data failed the assumption of equal variance, and a Kruskal-Wallis ANOVA on Ranks was performed. The p-value for those regions are derived from the Kruskal-Wallis ANOVA on Ranks, while the mean ± SEM were taken from the results of the one-way ANOVA. Abbreviations: rLSD = rostral dorsal LS; rLSV = rostral ventral LS; mLSV = medial ventral LS; cLSV = caudal ventral LS; mPVN = medial PVN; lPVN = lateral PVN; cPVN = caudal PVN; CeA = central amygdala; PAG = periaqueductal gray; lPAG = lateral periaqueductal gray.</p

Images from a near infrared western blot and immunohistochemical staining using an anti-NT antibody.

<p>(A) Bands were visualized at approximately 19 kDa, which corresponds with the molecular weight of the neurotensin/neuromedin N precursor protein, and at approximately 45 kDa. The identity of the protein weighing 45 kDa is unknown. (B) Example of the normalization method used to control for differences in background labeling. Background staining was relatively low across all sections analyzed (black square and top right panel), and was subtracted from the densitometric mean of regions of interest, such as the vLSM (white square and bottom right panel) in order to normalize brain sections analyzed. (C–D) NT staining was found in fibers and cell bodies (D, black arrows), which was expected. Regions labeled for NT were consistent with past immunohistochemical studies, suggesting that the antibody is specific to NT.</p

Primers for genes of interest and reference genes used for real-time qPCR.

<p>Primers for genes of interest and reference genes used for real-time qPCR.</p