Ghrelin's orexigenic action in the lateral hypothalamic area involves indirect recruitment of orexin neurons and arcuate nucleus activation

Abstract

Objective: Ghrelin is a potent orexigenic hormone, and the lateral hypothalamic area (LHA) has been suggested as a putative target mediating ghrelin's effects on food intake. Here, we aimed to investigate the presence of neurons expressing ghrelin receptor (a.k.a. growth hormone secretagogue receptor, GHSR) in the mouse LHA (LHA^GHSR neurons), its physiological implications and the neuronal circuit recruited by local ghrelin action.

Methods: We investigated the distribution of LHA^GHSR neurons using different histologic strategies, including the use of a reporter mice expressing enhanced green fluorescent protein under the control of the GHSR promoter. Also, we investigated the physiological implications of local injections of ghrelin within the LHA, and the extent to which the orexigenic effect of intra-LHA-injected ghrelin involves the arcuate nucleus (ARH) and orexin neurons of the LHA (LHA^orexin neurons) RESULTS: We found that: 1) LHA^GHSR neurons are homogeneously distributed throughout the entire LHA; 2) intra-LHA injections of ghrelin transiently increase food intake and locomotor activity; 3) ghrelin's orexigenic effect in the LHA involves the indirect recruitment of LHA^orexin neurons and the activation of ARH neurons; and 4) LHA^GHSR neurons are not targeted by plasma ghrelin.

Conclusions: We provide a compelling neuroanatomical and functional characterization of LHA^GHSR neurons in male mice that indicates that LHA^GHSR cells are part of a hypothalamic neuronal circuit that potently induces food intake.

Keywords: AgRP neurons; Food intake; GHSR; LHA.

Figure 1.. Neuroanatomical analysis of LHA^GHSR neurons.

A-D display representative images of coronal brain slices containing a set of hypothalamic regions including the LHA. Each panel shows GHSR-expressing cells through diverse techniques: A. ISHH against GHSR-mRNA in WT mice. B. ISHH against GHSR-mRNA in WT mice. This image was obtained from the Allen Mouse Brain Atlas. C. Immunofluorescence against eGFP in GHSR^eGFP mice. D. ISHH against GHSR-mRNA and immunostaining against eGFP in GHSR^eGFP mice. E display three representative high magnification images of LHA^eGFP+ cells with two or three branched fibers, obtained from GHSR^eGFP mice. F display a bar graph indicating a quantitative analysis of the number of eGFP+ cells along the LHA-rostro-caudal axis of GHSR^eGFP mice. Data is presented as mean ± SEM. In all cases, the wedges in the images indicate LHA^GHSR+ cells detected by each methodology. Insets show high magnification images taken from the areas indicated in low magnification photomicrographs. Scale bar: low magnification, 100 μm; high magnification 10 μm.

Figure 2.. Spatial reconstruction of LHA^eGFP+ cells in GHSR^eGFP mice.

The figure displays representative projection of all eGFP+ cells found in 54 serial section of GHSR^eGFP mouse. A-D shows different 3D-renderings of the LHA^eGFP+ cells, and surrounding structures. A and B displays a fronto-lateral view. C and D displays a frontal orthogonal view and a dorsal orthogonal view, respectively. The insets in B-D provide a wider view of each rendering. A, anterior, P posterior, D dorsal, V ventral, L left, R right, cc corpus callosum, fx fornix, mt mammillothalamic tracts, v ventricles system, opt optic tracts, LHA^eGFP+ cells.

Figure 3.. Intra-LHA-injected ghrelin transiently increases food intake and locomotor activity.

A displays representative images of a brain section belonging to a mouse subjected to an intra-LHA cannulation. The left image shows a schematic representation of the placement of the guide cannula and the internal cannula in a coronal brain section including the LHA. The right image shows a brain section including the trace of the internal cannula indicated by the punctuated red rectangles. B displays representative images of the hypothalamus of a mouse intra-LHA-injected with Fr-ghrelin (n=3). Insets display high magnification images of the areas indicated in low magnification images. C displays the quantitative analysis of the food intake induced by the injection of vehicle (n=5) or ghrelin (n=6) intra-LHA along 2-h after injection. Two-way ANOVA [F_int(2,27)=3.598 p=0.0412, F_time(2,27)=3.078 p=0.0623, F_treatment(1,27)=10.41 p=0.0033] followed by Tukey’s multiple comparisons test (**, p < 0.01 vs. vehicle intra-LHA). D displays the quantitative analysis of the distance travelled induced by intra-LH- injected vehicle (n=8) or ghrelin (n=11). Two-way ANOVA [F_int(30,450)=2.651 p<0.0001, F_time(30,450)=12.45 p<0.0001, F_treatment(1,15)=1.815 p=0.1979] followed by Tukey’s multiple comparisons test (**, p< 0.01 vs. vehicle intra-LHA). In this case, the graph displays only the first 15 min of activity after the injection. E-H indicate the quantitative analysis of the time that mice display digging (D), rearing (E), jumping (F) and grooming (G) along 30 min after the injection. I displays the quantitative analysis of the energy expenditure (VO₂) of the mice along 30 min after the injection (n=10). In all cases data is presented as mean ± SEM. Scale bar: low magnification, 100 μm; high magnification 10 μm.

Figure 4.. Intra-LHA-injected ghrelin induces c-Fos in LHA^orexin neurons and induces food intake and c-Fos in ARH^NPY/AgRP neurons via an orexin 1 receptor mechanism.

A-C display representative images of the ARH, VTA and LHA, respectively, of WT mice intra-LHA-injected with vehicle (n=8) or ghrelin (n=11) and subjected to immunostaining against c-Fos (black) and TH (brown). Inserts display high magnification images taken from areas indicated in low magnification images. Wedges point to TH+ cells and arrowheads point c-Fos+ cells. D display the quantitative analysis of the total food consumed after 2-h of the ghrelin or vehicle intra-LHA injection in mice with the ARH-intact or the ARH-ablated. Two-way ANOVA [F_int(1,15)=10.77 p=0.0050, F_PBS/MSG(1,15)=5.368 p=0.0351, F_veh/ghr(1,15)=19.67 p=0.0005] followed by Tukey’s multiple comparisons test (**, p< 0.01; ***, p<0.001). E displays representative images of brain sections that include the LHA subjected to immunostaining against c-Fos (black) and orexin (brown) proceeding from WT mice injected with vehicle or ghrelin intra-LHA. Arrows point to c-Fos+/orexin+ cells, wedges point to orexin+ cells and arrowheads point to c-Fos+ cells. F displays a representative image of the ARH of a NPY^hrGFP mouse subjected to immunostaining against orexin (red). Wedges indicate signal consistent with orexin+ fibers. G displays representative images of brain sections that include the ARH subjected to immunostaining against c-Fos (purple) proceeding from NPY^hrGFP mice injected with vehicle or ghrelin intra-LHA. Arrows point to c-Fos+/hrGFP+ cells and wedges point to hrGFP+ cells. In all cases, insets display high magnification images taken from the areas indicated in low magnification photomicrographs. H displays the quantitative analysis of the total food consumed after 2 h of the vehicle or ghrelin intra-LHA injection in NPY^hrGFP mice IP preinjected with SB-334867 or vehicle. Two-way ANOVA [F_int(1,14)=7.055 p=0.0188, F_SB/Veh(1,14)=12.72 p=0.0031, F_veh/ghr(1,14)=12.32 p=0.0035] followed by Tukey’s multiple comparisons test (**, p< 0.01; ns, non-significative). I display the quantitative analysis of the fraction of hrGFP cells in the ARH nucleus of the NPY^hrGFP mice that are c-Fos-IR. Two-way ANOVA [F_int(1,14)=9.089 p=0.0093, F_veh/SB(1,14)=8.005 p=0.0134, F_veh/ghr(1,14)=24.91 p=0.0002] followed by Tukey’s multiple comparisons test (**, p< 0.01; ***, p<0.001; ns, non-significative). In all cases, data is presented as mean ± SEM. Scale bar: low magnification, 100 μm; high magnification 10 μm. Quantitative analysis corresponding to panels A-C and E can be found in the supplementary figure 2.

Figure 5.. A fraction of LHA^GHSR cells are nNOS+ or neurotensin+.

A displays immunostaining against eGFP and orexin in the LHA of GHSR^eGFP mice. B displays an ISHH against GHSR-mRNA and immunostaining against orexin in the LHA of WT mice. C-J display immunostaining against eGFP and diverse neuropeptides in the LHA of GHSR^eGFP mice including nNOS, MCH, parvalbumin, oxytocin, neurotensin, somatostatin, TRH and galanin respectively. K displays immunostaining against eGFP in GHSR^eGFP/Gad2^tdTomato mice. Insets display high magnification images taken from the areas indicated in low magnification photomicrographs. Arrows indicate double IR-cells (eGFP+/nNOS+ or eGFP+/neurotensin+ cells), wedges indicate eGFP+ cells and arrowheads indicate positive cells for the different neurochemical markers. Scale bar: low magnification, 100 μm; high magnification 10 μm.

Figure 6.. Circulating ghrelin do not induce c-Fos in LHA^GHSR cells and Fr-ghrelin do not reach the LHA.

A display representative images of brain sections containing the LHA subjected to immunostaining against c-Fos and eGFP from GHSR^eGFP mice systemically (n=4) or ICV-injected (n=6) with ghrelin or vehicle (n=6). Arrows indicate c-Fos+/eGFP+ cells, wedges indicate eGFP+ cells and arrowheads indicate c-Fos+ cells. Kruskal-Wallis test [H(3,13)=6.087 p=0.0206] followed by Dunn’s multiple comparisons test (p=0.0227 ICV-injected ghrelin vs. vehicle). B displays representative images if brain section containing the LHA and the ARH from WT mice systemically (n=3) or ICV-injected (n=6) with Fr-ghrelin or vehicle (n=4). Wedges indicate Fr-ghrelin+ cells. One-way ANOVA [F(2,12)=0.7483 p=0.4940]. C display representative images of brain section containing the LHA subjected to immunostaining against c-Fos and eGFP from of GHSR^eGFP mice exposed to calorie restriction (n=7) or fed ad libitum (n=4). T-test [t(9)=0.8666 p=0.4087]. Wedges indicate eGFP+ cells and arrowheads indicate c-Fos+ cells. In all cases, insets display high magnification images taken from the areas indicated in low magnification photomicrographs. Scale bar: low magnification, 100 μm; high magnification 10 μm.