Central nesfatin-1-expressing neurons are sensitive to peripheral inflammatory stimulus

Abstract

Recently, a novel factor with anorexigenic properties was identified and called nesfatin-1. This protein (82 aac) is not only expressed in peripheral organs but it is also found in neurons located in specific structures including the hypothalamus and the brainstem, two sites strongly involved in food intake regulation. Here, we studied whether some of the neurons that become activated following an injection of an anorectic dose of lipopolysaccharides (LPS) exhibit a nesfatin-1 phenotype. To this end, we used double immunohistochemistry to target the expression of the immediate-early gene c-fos and of nesfatin-1 on coronal frozen sections of the rat brain. The number of c-Fos+/nesfatin-1+ neurons was evaluated in the immunosensitive structures reported to contain nesfatin-1 neurons; i.e. paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SON), arcuate nucleus (ARC) and nucleus of the solitary tract (NTS). LPS strongly increased the number of c-Fos+/nesfatin-1+ neurons in the PVN, SON and NTS, and to a lesser extent in the ARC. Triple labeling showed that a portion of the nesfatin-1 neurons activated in response to LPS within the NTS are catecholaminergic since they co-express tyrosine hydroxylase (TH). Our data therefore indicate that a portion of nesfatin-1 neurons of both the hypothalamus and brainstem are sensitive to peripheral inflammatory signals, and provide the first clues suggesting that centrally released nesfatin-1 may contribute to the neural mechanisms leading to endotoxaemic anorexia.

Figure 1

A-F: Representative photomicrographs of c-Fos immunoreactivity in coronal sections of the PVN, ARC and SON. Images derived from saline-injected (A-C) and LPS-treated (D-F) rats. G-L: Analysis of c-Fos and nesfatin-1 (green) double-labeling in coronal sections through the hypothalamus of LPS-treated rats. Dashed boxes in low magnification images (G-I) indicate the area where high magnification images (J-L) originate. Arrowheads: c-Fos+/Nesf+ neurons; arrows: c-Fos+ neurons negative for nesfatin-1. ARC arcuate nucleus; ox, optic chiasm; PVN, paraventricular nucleus of the hypothalamus; SON: supraoptic nucleus; 3V, third ventricle. Scale bars: 200 μm (A-I), 50 μm (J-L).

Figure 2

A-B: c-Fos immunoreactivity within the DVC. Representative coronal sections of the DVC of saline-injected (A) and LPS-treated (B) rats. C-D: c-Fos and nesfatin-1 (green) double-labeling in coronal sections through the DVC of LPS-treated rats. D: High magnification of c-Fos/nesfatin-1 positive neurons in the NTS. Dashed box in C indicates the area where high magnification image originates. Arrowheads: c-Fos+/Nesf+ neurons; arrow: c-Fos+ neuron negative for nesfatin-1. E: Quantification of the number of c-Fos and c-Fos/nesfatin-1 immunoreactive cells at the NTS subpostremal level of rats injected with either saline or LPS solution. Starred values are significantly different from saline-injected animals, *** p < 0.001. AP, area postrema; cc, central canal; DMNX, dorsal motor nucleus of the vagus; NTS, nucleus of the solitary tract; ts, tractus solitarius. Scale bars: 100 μm (A-C), 50 μm (D).

Figure 3

Quantification of the number of c-Fos and c-Fos/nesfatin-1 immunoreactive cells in the PVN, SON and ARC after an i.p. injection of either saline or LPS solution. Starred values are significantly different from saline-injected rats, ** p < 0.01; *** p < 0.001.

Figure 4

Representative photomicrographs of c-Fos (DAB precipitate)/nesfatin-1 (green)/TH (red) triple labeling performed on brainstem coronal sections of rats treated with LPS. Note the presence of c-Fos positive neurons co-expressing nesfatin-1 and TH (arrowheads), c-Fos+/Nesf+ neuron negative for TH (arrow) and c-Fos+/Nesf-/TH-neuron (asterisk). Dashed box in image (A) indicates the area where high magnification images (D-E) originate. AP, area postrema; cc, central canal; DMNX, dorsal motor nucleus of the vagus; NTS, nucleus of the solitary tract; ts, tractus solitarius. Scale bars: 150 μm (A-C); 40 μM (D-F).