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. 2016 Jun 27:10:289.
doi: 10.3389/fnins.2016.00289. eCollection 2016.

Action of Administered Ciliary Neurotrophic Factor on the Mouse Dorsal Vagal Complex

Martina Senzacqua  1 , Ilenia Severi  1 , Jessica Perugini  1 , Samantha Acciarini  1 , Saverio Cinti  2 , Antonio Giordano  1
Affiliations

Action of Administered Ciliary Neurotrophic Factor on the Mouse Dorsal Vagal Complex

Martina Senzacqua et al. Front Neurosci. .

Abstract

Ciliary neurotrophic factor (CNTF) induces weight loss in obese rodents and humans through activation of the hypothalamic Jak-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway. Here, we tested the hypothesis that CNTF also affects the brainstem centers involved in feeding and energy balance regulation. To this end, wild-type and leptin-deficient (ob/ob and db/db) obese mice were acutely treated with intraperitoneal recombinant CNTF. Coronal brainstem sections were processed for immunohistochemical detection of STAT3, STAT1, STAT5 phosphorylation and c-Fos. In wild-type mice, CNTF treatment for 45 min induced STAT3, STAT1, and STAT5 phosphorylation in neurons as well as glial cells of the area postrema; here, the majority of CNTF-responsive cells activated multiple STAT isoforms, and a significant proportion of CNTF-responsive glial cells bore the immaturity and plasticity markers nestin and vimentin. After 120 min CNTF treatment, c-Fos expression was intense in glial cells and weak in neurons of the area postrema, it was intense in several neurons of the rostral and caudal solitary tract nucleus (NTS), and weak in some cholinergic neurons of the dorsal motor nucleus of the vagus. In the ob/ob and db/db mice, Jak-STAT activation and c-Fos expression were similar to those induced in wild-type mouse brainstem. Treatment with CNTF (120 min, to induce c-Fos expression) and leptin (25 min, to induce STAT3 phosphorylation) demonstrated the co-localization of the two transcription factors in a small neuron population in the caudal NTS portion. Finally, weak immunohistochemical CNTF staining, detected in funiculus separans, and meningeal glial cells, matched the modest amount of CNTF found by RT-qPCR in micropunched area postrema tissue, which in contrast exhibited a very high amount of CNTF receptor. Collectively, the present findings show that the area postrema and the NTS exhibit high, distinctive responsiveness to circulating exogenous and, probably, endogenous CNTF. VSports手机版.

Keywords: Stat signaling; area postrema; brainstem; c-Fos; cholinergic; dorsal motor nucleus of the vagus; nestin; solitary tract nucleus. V体育安卓版.

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Figures (VSports)

Figure 1
Figure 1
P-STAT immunohistochemistry in coronal sections of mouse area postrema. After 45 min CNTF treatment, P-STAT3 (A), P-STAT1 (B), and P-STAT5 (C) immunoreactivity was detected in the nucleus of many cells of the area postrema (AP). Insets: the area postrema of control mice processed for immunohistochemistry against the three P-STAT isoforms. Double-staining and confocal microscopy experiments in a CNTF-treated mouse (D–F): P-STAT3 staining is ubiquitous and is also found in the funiculus separans (fs), whereas P-STAT1 staining is detected in a smaller number of cells, most of which lie in the central portion of the area postrema. At higher magnification, the majority of P-STAT3-positive cells also express P-STAT1 (G–I, arrows) or P-STAT5 (J–L), arrows. Panels (G–I) are enlargements of the areas framed in Panels (D–F), respectively. Bar: (A–F), 120 μm; insets of (A–C), 300 μm; (G–I), 25 μm; (J–L), 18 μm.
Figure 2
Figure 2
Phenotypic characterization of CNTF-responsive cells in the mouse area postrema. Double-staining and confocal microscopy experiments showed that in mice treated with CNTF for 45 min some P-STAT3-positive cells co-localized with the neuronal marker HuC/D (A–C, arrows), the intermediate filament proteins nestin (D–F, arrows) or vimentin (G–I, arrows), or the glial marker GFAP (J–L, arrows). Bar:(A–C), 12 μm; (D–L), 8 μm.
Figure 3
Figure 3
Phenotypic characterization of CNTF-responsive neurons in the mouse area postrema. Double-staining and confocal microscopy experiments showed that in mice treated with CNTF for 45 min a few P-STAT3-positive cells were positive for dopamine-beta-hydroxylase (DBH; A–C, arrows) or tryptophan hydroxylase (TPH; D–F, arrows). In contrast, glutamic acid decarboxylase 67 (GAD67; G–I, arrowheads) and choline acetyltransferase (ChAT; J–L, arrowheads) expressing neurons never co-localized with P-STAT3. In a mouse treated with CNTF for 80 min (M–P), some P-STAT3-positive cells also expressed c-Fos (arrows), whereas the P-STAT3 HuC/D-positive neurons did not (arrowheads). Bar: (A–C), 10 μm; (D–L), 12 μm; (M–P), 18 μm.
Figure 4
Figure 4
P-STAT3 and c-Fos immunohistochemistry in coronal brainstem sections from mice treated with CNTF for 120 min. Double-staining and confocal microscopy experiments (A–C) still showed some P-STAT3-positive cells in the area postrema, including the funiculus separans (fs), where CNTF-responsive cells are positive for GFAP (insets, arrows). Immunoperoxidase histochemistry showed nuclear c-Fos expression in numerous cells of the rostral (D) and caudal (E) portions of the solitary tract nucleus (NTS) and in the area postrema (E, AP). 4V, fourth ventricle. Double-staining experiments demonstrated that in the area postrema (F–H) only few HuC/D-positive neurons expressed weak c-Fos staining (arrows), whereas in the NTS (I–K) all c-Fos-positive cells were HuC/D-positive neurons (arrows). Insets of (A–C) are enlargements of the corresponding framed areas. In (D) and (E), insets on the left show the corresponding structures from a control mouse processed for c-Fos immunohistochemistry; those on the right show the adjacent Nissl-stained section. Bar: (A–C), 150 μm; insets of (A–C), 20 μm; (D) and (E), 150 μm; insets of (D) and (E), 400 μm; (F–K), 10 μm.
Figure 5
Figure 5
c-Fos activation in brainstem cholinergic neurons by CNTF. Double-staining and confocal microscopy experiments after 120 min CNTF treatment showed c-Fos expression (B) in cells of the area postrema (AP), solitary tract nucleus (NTS) and dorsal motor nucleus of the vagus (DMX), depicted by immunofluorescent detection of choline acetyltransferase (ChAT,A), the marker of cholinergic pre-ganglionic vagal neurons. Panels (D–F) are the enlargements of the corresponding areas framed in (A–C), showing two large DMX cholinergic neurons expressing c-Fos (arrows). Some small cholinergic neurons of the NTS were also c-Fos-positive (G–I, arrows). Sol, solitary tract. Bar: (A–C), 50 μm; (D–F), 10 μm; (G–I), 25 μm.
Figure 6
Figure 6
Relationship between CNTF and leptin signaling in the mouse brainstem. CNTF treatment for 45 min induced STAT3 (A,E), STAT1 (B,F), and STAT5 (C,G) phosphorylation in the area postrema of ob/ob (A–D) and db/db (E–H) obese mice and c-Fos expression (D,H) in the area postrema (AP) and solitary tract nucleus (NTS). Treatment with leptin for 25 min (J) activated STAT3 signaling in neurons and fibers in the caudal portion of the NTS, enlarged in the inset. (I) corresponding structures from a control mouse processed for P-STAT3 immunohistochemistry. Inset of I shows the adjacent Nissl-stained section. Double-staining and confocal microscopy experiments in a coronal brainstem section from a mouse treated for 120 min with CNTF to induce c-Fos expression and with leptin for 25 min to induce STAT3 activation (K–M) show some NTS neurons (enlarged in the insets, arrows) expressing both c-Fos and P-STAT3. Insets of (J) and (K–M) are enlargements of the corresponding framed areas. Bar:(A–C) and (E–G), 150 μm; (D, H–J,) and (K–M), 120 μm; insets of (J) and (K–M), 30 μm; inset of (I), 300 μm.
Figure 7
Figure 7
CNTF and CNTFRα expression in the mouse area postrema. Peroxidase immunohistochemistry demonstrated CNTF immunoreactivity in small portions of the funiculus separans (A, fs) and of the meninges (A,B, arrowheads). The antibody mainly stained cellular projections (left-side insets, arrowheads) and rarely cell somata (left-side inset in B, arrow). In A and B, insets on the left show the enlargements of the corresponding framed areas, whereas those on the right show the corresponding structures processed for CNTF immunohistochemistry from a CNTF-knockout mouse. Double-labeling experiments (C–E) showed that CNTF-immunoreactive structures were positive for the GFAP (arrows). RT-PCR demonstrated that in hypothalamic specimens CNTFRα mRNA was 264.94 times higher than that of the CNTF (F), whereas in the brainstem samples it was 424.89 times higher than that of its ligand (G). Bar: (A,B), 100 μm; left insets of (A,B), 60 μm; right insets of (A,B), 300 μm; (C–E), 50 μm.
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