Distribution of Growth Hormone Responsive Neurons in the Mouse Hypothalamus

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 88-108-GHRH, GH & IGF Biology & Signaling
Basic/Translational
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-106
Ilona C. Kokay*1, Nicola H. Guy2, Paul R. Le Tissier3 and David R. Grattan1
1University of Otago, Dunedin, New Zealand, 2University of Otago, 3UCL, London, United Kingdom
Growth hormone (GH) receptors have been identified in the brain, but the pattern of activation of neurons activated by peripheral GH has not been reported. An important downstream signalling pathway activated by the binding of GH to the growth hormone receptor is the Jak-STAT pathway. Upon binding of GH to its receptor, Stat proteins, including STAT5, are phosphorylated and translocate to the nucleus to influence gene transcription (pSTAT5). The aim of this study was to measure GH-induced pSTAT5 in the brain by immunohistochemistry to provide a detailed mapping of GH responsive neurons in the mouse brain.  Adult male C57B6 mice, aged 10-12 weeks, were injected intraperitoneally with 25 ug of recombinant mouse growth hormone (NHPP) (n=10), or vehicle control (n=8).  Forty-five minutes later, mice were transcardially perfused with 4% paraformaldehyde, and brains collected and cryoprotected in a 30% sucrose solution overnight.  All animals were treated with bromocriptine subcutaneously (100 ug,) 24, 12 and 2 hours before perfusion, to eliminate endogenous prolactin secretion that is also known to activate STAT5 in the brain.  Brains were then frozen and serial 30 µm coronal brain sections were cut through the hypothalamus. Free-floating sections were subjected to an antigen retrieval step, incubated with pSTAT5 primary antibody (tyr694, Cell Signalling) for 48 hours at 4°C, followed by incubation with goat anti-rabbit biotinylated secondary antibody for 90 mins. After an avidin-biotin-peroxidase step, labeled cells were visualized using nickel DAB as the substrate.  Positively-labelled cells were counted using ImageJ software (NIH). GH-induced pSTAT5 was identified throughout the hypothalamus. The highest levels of pSTAT5-labelled cells were seen in the arcuate nucleus, the medial preoptic nucleus, the anterior-ventral preoptic nucleus and the paraventricular nucleus.  Staining was also pronounced in the periventricular region at the level of the paraventricular nucleus. In these same sections, pSTAT5 positive cells were also observed outside the hypothalamus, including the amygdala and the bed nucleus of the stria terminalis. As the distribution of GH-induced pSTAT5 was similar to that previously observed for prolactin-induced pSTAT5, we have also examined the GH response in transgenic mice that lack prolactin receptors.  Interestingly, GH-induce pSTAT5 was significantly reduced in these mice. The data provide important neuroanatomical information to identify GH-responsive networks in the brain.

Nothing to Disclose: ICK, NHG, PRL, DRG

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