Synaptic input organization of the melanocortin system predicts diet-induced hypothalamic reactive gliosis and obesity

Abstract

The neuronal circuits involved in the regulation of feeding behavior and energy expenditure are soft-wired, reflecting the relative activity of the postsynaptic neuronal system, including the anorexigenic proopiomelanocortin (POMC)-expressing cells of the arcuate nucleus. We analyzed the synaptic input organization of the melanocortin system in lean rats that were vulnerable (DIO) or resistant (DR) to diet-induced obesity. We found a distinct difference in the quantitative and qualitative synaptology of POMC cells between DIO and DR animals, with a significantly greater number of inhibitory inputs in the POMC neurons in DIO rats compared with DR rats. When exposed to a high-fat diet (HFD), the POMC cells of DIO animals lost synapses, whereas those of DR rats recruited connections. In both DIO rats and mice, the HFD-triggered loss of synapses on POMC neurons was associated with increased glial ensheathment of the POMC perikarya. The altered synaptic organization of HFD-fed animals promoted increased POMC tone and a decrease in the stimulatory connections onto the neighboring neuropeptide Y (NPY) cells. Exposure to HFD was associated with reactive gliosis, and this affected the structure of the blood-brain barrier such that the POMC and NPY cell bodies and dendrites became less accessible to blood vessels. Taken together, these data suggest that consumption of an HFD has a major impact on the cytoarchitecture of the arcuate nucleus in vulnerable subjects, with changes that might be irreversible due to reactive gliosis.

Fig. 1.

The metabolic phenotype of DR and DIO rats fed an SD and those fed an HFD. (A) Body weight gain. (B) Fat mass. a: P < 0.05. (C) Insulin level. a: P < 0.05. (D) Leptin level. a: P < 0.05. (E) Total ghrelin level. a: P < 0.05. All results are mean ± SEM.

Fig. 2.

Synaptic input organization of DR and DIO rats. (A) Bar graphs showing the numbers of perikaryal symmetrical and asymmetrical connections on POMC neurons of DR and DIO animals fed an SD. a and b: P < 0.05. (B and C) Representative electron micrographs showing asymmetrical, putative stimulatory (+; B), and, symmetrical, putative inhibitory (−; C) synapses taken from POMC-immunolabeled perikarya of an SD-fed DR rat (B) and of an SD-fed DIO rat (C). (Scale bar in B: 1 μm for B, C, E, and F.) (D) Bar graphs showing the numbers of perikaryal symmetrical and asymmetrical connections on POMC neurons of DR and DIO animals fed an HFD. (E and F) Representative electron micrographs showing POMC-labeled perikarya of a DR HFD mouse (E) and a DIO HFD mouse (F). Note that instead of the axon terminals (A) in the DR HFD POMC cells, there is glial ensheathment (indicated by green pseudocolor) of the POMC perikarya of the DIO HFD animal. Bar graphs represent mean ± SEM. (G) Bar graphs indicating the total numbers of synapses on POMC perikarya of DR and DIO rats fed an SD and an HFD. a: P < 0.05 DR HFD versus DR SD; b: P < 0.05 DIO SD versus DR SD; c: P < 0.05 DIO HFD versus DIO SD. (H) Bar graphs indicating glial ensheathment on POMC perikarya of DR and DIO animals fed an HFD. a: P < 0.05.

Fig. 3.

Synaptology of arcuate nucleus NPY and POMC neurons from SD- and HFD-fed DIO mice. (A and B) Bar graphs showing total, inhibitory, and excitatory synapses on POMC (A) and NPY (B) neurons from animals fed an SD and those fed an HFD. a: P < 0.05 HFD versus SD values. (C and D) Representative light micrographs showing GFAP immunolabeling in the arcuate nucleus of animals fed an SD (C) or an HFD (D). (Scale bar in E: 10 μm for C and D.) (E) Electron micrograph of a typical direct apposition between a POMC perikaryon and a vessel in SD-fed mice. (Inset) Higher-power magnification of a contact between a POMC perikaryon and the glia limitans of a vessel. The star indicates the space between the POMC perykaryon and the endothelial cell of a vessel. Arrows point to the thin layer of glia. (Scale bar: 1 μm.) (F and G) The same electron micrograph of a GFP-NPY perikaryon from a HFD-fed animal in the vicinity of a vessel in the arcuate nucleus. Note the increased presence of glia between the vessel and the labeled perikaryon (arrows in F) and other parts of the cell body. The pseudocolor in G indicates glial processes around the labeled NPY cell. (Scale bar in F: 1 μm for F and G.) Bar graphs represent mean ± SEM.

Fig. 4.

Capillaries and arcuate nucleus transcript levels of GFAP, POMC, and NPY from mice fed an SD and an HFD. (A and B) Low-power magnification electron micrographs showing capillaries (black arrows) in the arcuate nucleus from an animal fed an SD (A) and an animal fed an HFD (B). (Scale bar in A: 10 μm for A and B.) (C) Bar graphs showing no statistically significant differences in numbers of capillaries between the two groups. (D) Bar graph showing that the diameter of capillary lumen is greater in the arcuate nucleus of animals fed an HFD compared with animals fed an SD. (E) Graph showing an increased level of GFAP mRNA in the arcuate nucleus of HFD-fed mice compared with SD-fed lean control mice (a: P < 0.05). (F) Graph showing an increased level of POMC mRNA in the arcuate nucleus of HFD-fed mice compared with SD-fed lean controls (a: P < 0.05). (G) Graph showing a decreased level of NPY mRNA in the arcuate nucleus of HFD-fed DIO mice compared with SD-fed lean controls (a: P < 0.05). Bar graphs represent mean ± SEM.