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. 2012;7(2):e31462.
doi: 10.1371/journal.pone.0031462. Epub 2012 Feb 20.

Ghrelin indirectly activates hypophysiotropic CRF neurons in rodents

Agustina Cabral  1 , Olga Suescun, Jeffrey M Zigman, Mario Perello
Affiliations

Ghrelin indirectly activates hypophysiotropic CRF neurons in rodents

Agustina Cabral et al. PLoS One. 2012.

Abstract

Ghrelin is a stomach-derived hormone that regulates food intake and neuroendocrine function by acting on its receptor, GHSR (Growth Hormone Secretagogue Receptor). Recent evidence indicates that a key function of ghrelin is to signal stress to the brain. It has been suggested that one of the potential stress-related ghrelin targets is the CRF (Corticotropin-Releasing Factor)-producing neurons of the hypothalamic paraventricular nucleus, which secrete the CRF neuropeptide into the median eminence and activate the hypothalamic-pituitary-adrenal axis. However, the neural circuits that mediate the ghrelin-induced activation of this neuroendocrine axis are mostly uncharacterized. In the current study, we characterized in vivo the mechanism by which ghrelin activates the hypophysiotropic CRF neurons in mice. We found that peripheral or intra-cerebro-ventricular administration of ghrelin strongly activates c-fos--a marker of cellular activation--in CRF-producing neurons. Also, ghrelin activates CRF gene expression in the paraventricular nucleus of the hypothalamus and the hypothalamic-pituitary-adrenal axis at peripheral level. Ghrelin administration directly into the paraventricular nucleus of the hypothalamus also induces c-fos within the CRF-producing neurons and the hypothalamic-pituitary-adrenal axis, without any significant effect on the food intake. Interestingly, dual-label immunohistochemical analysis and ghrelin binding studies failed to show GHSR expression in CRF neurons. Thus, we conclude that ghrelin activates hypophysiotropic CRF neurons, albeit indirectly.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Ghrelin administration activates CRF neurons of the PVN.
Panels A to D show representative photomicrographs of brains sections subjected to double immunohistochemistry using anti-CRF (brown staining) and anti-c-fos (purple-black staining) antiserum. Upper panels show the ARC and bottom panels show the PVN. Images of panel A and C are from a vehicle-treated mouse and images of panel B and D from a ghrelin-treated mouse. Inserts in PVN images show high magnification of areas marked in low magnification images. Arrows point to dual-labeled cells. Scale bars, 100 µm (low magnification), 10 µm (high magnification). Panels E and F show schematic drawings of middle level of the PVN of a vehicle- and ghrelin-treated mouse, respectively. Filled circles represent CRF-IR neurons positive for c-fos, and open circles indicate single-labeled CRF-IR cell bodies. Gray letters indicate the PVN subdivisions: P: periventricular; M: medial; C: compact.
Figure 2
Figure 2. ICV ghrelin administration activates CRF neurons of the PVN.
Panels show representative photomicrographs of brains sections subjected to double immunohistochemistry using anti-CRF (brown staining) and anti-c-fos (purple-black staining) antiserum. Upper panels show the ARC and bottom panels show the PVN. Images of panel A and C are from a vehicle-treated mouse and images of panel B and D from a ghrelin-treated mouse. Inserts in PVN images show high magnification of areas marked in low magnification images. Arrows point to dual-labeled cells. Scale bars, 100 µm (low magnification), 10 µm (high magnification).
Figure 3
Figure 3. ICV ghrelin administration activates the HPA axis.
Panel A shows comparative values of RT-qPCR for CRF mRNA in the PVN micro-dissected punches obtained from vehicle-treated and ghrelin-treated mice sacrificed at different time points. Data is shown as mRNA levels of CRF relative to the housekeeping gene Cyclophilin A, calculated by the comparative threshold cycle (Ct) method. Panel B shows comparative values of specific ELISA for plasma corticosterone obtained from vehicle- and ghrelin-treated mice sacrificed at different time points. Data represent the mean±SEM. *p≤0.05. **p≤0.01. ***p≤0.001.
Figure 4
Figure 4. Ghrelin can be microinjected exclusively into the PVN.
Panel A shows a schematic diagram of a coronal section of the mouse brain with the localization of a correctly implanted cannula (black). This experimental strategy allowed us to administer vehicle, containing or not ghrelin, 500 µm above the PVN (blue) via the injector cannula (gray). Panel B shows a representative photomicrograph of a coronal brain section subjected to thionin staining of an intra-PVN injected mouse. Lines label injector cannula tracts, where no thionin staining is observed. Panels C, D and E show representative photomicrographs of brain sections, containing the PVN, subjected to immunohistochemistry using anti-c-fos (purple-black staining) antiserum in “saline-treated”, “ghrelin-hits” and “ghrelin-misses” groups, respectively. Scale bars, 100 µm.
Figure 5
Figure 5. Ghrelin microinjection into the PVN activates CRF neurons.
Panels A to B show representative photomicrographs of brains sections subjected to double immunohistochemistry using anti-CRF (brown staining) and anti-c-fos (purple-black staining) antiserum. Upper panels show the ARC and bottom panels show the PVN. Images of panel A and C are from a vehicle-treated mouse and images of panel B and D from a ghrelin-treated mouse. Inserts in C and D panels show high magnification of areas marked in low magnification images. Arrows to dual-labeled cells. Scale bars, 100 µm (low magnification), 10 µm (high magnification).
Figure 6
Figure 6. Ghrelin microinjection into the PVN activates the HPA axis.
Panel A shows comparative values of RT-qPCR for CRF mRNA in the PVN micro-dissected punches obtained from intra-PVN ghrelin-treated, intra-PVN vehicle-treated and misses groups. Data is shown as mRNA levels of CRF relative to the housekeeping gene Cyclophilin A, calculated by the comparative threshold cycle (Ct) method. Panel B shows comparative values of specific ELISA for plasma corticosterone obtained from intra-PVN ghrelin-treated, intra-PVN vehicle-treated and misses groups. Data represent the mean±SEM. *p≤0.05.
Figure 7
Figure 7. Ghrelin binding to the compact part of the PVN is scarce.
This figure shows representative photomicrographs of brains sections subjected to a ghrelin binding assay (see Material and Methods for details), at low and high magnification (inserts). Purple/Black signal indicates specific binding of ghrelin. Panel A shows an internal positive control of the assay in which positive ghrelin binding to cells located in the ARC and to the ventrolateral subdivision of the VMH (vlVMH) is observed, when brain section are incubated with 1 µg/ml of ghrelin. Panel B shows the PVN level for a negative control, where no exogenous ghrelin was added. Panels C and D show positive ghrelin binding to cells located in the PVN, in brain section previously incubated with 0.1 or 1 µg/ml of ghrelin, respectively. Inserts in each image show high magnification of areas marked in low magnification images. Arrows point to labeled cells. Scale bars, 100 µm (low magnification), 10 µm (high magnification).
Figure 8
Figure 8. CRF-IR neurons of the PVN do not express GHSR.
Upper panels show representative ISHH photomicrographs of thionin counterstained coronal sections of brain mouse. Panels A and B show photomicrographs of the ARC and PVN, respectively. Black silver granules, representing the binding of GHSR antisense riboprobes, can be detected at a higher density in the ARC (A) and with smaller density in the PVN (B). Bottom panels show representative photomicrographs of ISHH for GHSR (black silver granules) and IHC for CRF (brown staining) on coronal brain sections of mice. Panels C and D show photomicrographs of the ARC and PVN, respectively. Inserts in each image show high magnification of areas marked in low magnification images. White arrows point to examples of cells labeled with GHSR riboprobe. Black arrowhead point to examples of cells labeled with anti-CRF IHC. Scale bars, 100 µm (low magnification), 20 µm (high magnification).
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