Neurochemical Characterization of Brainstem Pro-Opiomelanocortin Cells

Abstract

Genetic research has revealed pro-opiomelanocortin (POMC) to be a fundamental regulator of energy balance and body weight in mammals. Within the brain, POMC is primarily expressed in the arcuate nucleus of the hypothalamus (ARC), while a smaller population exists in the brainstem nucleus of the solitary tract (POMCNTS). We performed a neurochemical characterization of this understudied population of POMC cells using transgenic mice expressing green fluorescent protein (eGFP) under the control of a POMC promoter/enhancer (PomceGFP). Expression of endogenous Pomc mRNA in the nucleus of the solitary tract (NTS) PomceGFP cells was confirmed using fluorescence-activating cell sorting (FACS) followed by quantitative PCR. In situ hybridization histochemistry of endogenous Pomc mRNA and immunohistochemical analysis of eGFP revealed that POMC is primarily localized within the caudal NTS. Neurochemical analysis indicated that POMCNTS is not co-expressed with tyrosine hydroxylase (TH), glucagon-like peptide 1 (GLP-1), cholecystokinin (CCK), brain-derived neurotrophic factor (BDNF), nesfatin, nitric oxide synthase 1 (nNOS), seipin, or choline acetyltransferase (ChAT) cells, whereas 100% of POMCNTS is co-expressed with transcription factor paired-like homeobox2b (Phox2b). We observed that 20% of POMCNTS cells express receptors for adipocyte hormone leptin (LepRbs) using a PomceGFP:LepRbCre:tdTOM double-reporter line. Elevations in endogenous or exogenous leptin levels increased the in vivo activity (c-FOS) of a small subset of POMCNTS cells. Using ex vivo slice electrophysiology, we observed that this effect of leptin on POMCNTS cell activity is postsynaptic. These findings reveal that a subset of POMCNTS cells are responsive to both changes in energy status and the adipocyte hormone leptin, findings of relevance to the neurobiology of obesity.

Keywords: NTS; Pomc; leptin receptor; obesity.

Figure 1.

POMC^NTS expression and Pomc^eGFP validation. A: Adapted brain atlas (69) schematics of NTS (shaded green area) at -7.56 to -7.76 from bregma. B: In situ hybridization histochemistry (ISSH) with a ³⁵S-labelled Pomc riboprobe revealed the highest level of Pomc mRNA (black grains) within the NTS is between -7.5 to -7.8 from bregma in adult wild-type C57BL/6 mice (n = 7). These findings were reproduced in Pomc^eGFP mice (n = 7). C: Representative photomicrographs of GFP immunofluorescence (IF) in adult male and female Pomc^eGFP mice (n = 20) also identified that the most abundant distribution of GFP-IR cells is between -7.5 to -7.8 from bregma. D: Expression of Pomc mRNA in GFP cells in extracted NTS of Pomc^eGFP mice was confirmed using fluorescence-activating cell sorting (FACS) followed by qPCR analysis. Pomc and Gfp mRNA were normalized to that of the housekeeping gene Gapdh. Expression of Pomc and Gfp mRNA is expressed as a percentage of that determined in GFP-positive (GFP+) cells and compared to GFP-negative (GFP-) cells. CC, central canal, scale bar B 500 μm; scale bar C 125 μm.

Figure 2.

Neurochemical characterization of POMC^NTS neurons. Immunohistochemical analysis of adult male and female Pomc^eGFP mice demonstrated GFP-IR cells (green) to be negative for catecholamine cell marker tyrosine hydroxylase (TH)-IR (red) (A), nesfatin-IR (red) (B), acetylcholine cell marker choline acetyltransferase (ChAT)-IR (red) (C), protein seipin-IR (red) (D) and enzyme nitric oxide synthase 1 (nNOS)-IR (red) (n = 3–5 mice per analysis) (E). Dual IHC and ISHH analysis of adult male and female Pomc^eGFP mice demonstrated the absence of GFP-IR co-expression (brown cytoplasmic stain) with ³⁵S preproglucagon (Ppg) (F), ³⁵S cholecystokinin (Cck)(G) or ³⁵S brain derived neurotrophic factor (Bdnf) mRNA (black grains) (n = 3–5 mice per analysis) (H). I: Double-IF analysis in adult male and female Pomc^eGFP:LepRb^Cre:tdTomato mice (n = 9) revealed a subset of POMC-expressing neurons (green) were co-expressed with LepRb-expressing cells (red). J: In Pomc^eGFP mice (n = 4) and LepRb^CRE:eGFP mice (n = 4) (K), all GFP-IR cells (green) were Phox2b-IR positive (red). L: Schematic illustrating overlap of POMC, Phox2b, and LepRb in the NTS. Level of NTS presented in (A–K), -7.56 to -7.76 from bregma. Arrows represent dual-labeled cells. CC, central canal; scale bar, 50 μm applies to (A–K).

Figure 3.

Pomc ^eGFP cells increase their activity in response to food intake. A-D:Pomc^eGFP mice exhibit more FOS-IR in GFP-IR cells within the NTS in response refeeding compared to cells from mice in the ad libitum–fed or 12-hour fasted state (n = 4–5). E: Endogenous plasma leptin levels are increased following refeeding (n = 8) compared to ad libitum–fed (n = 9) or 12-hour fasted mice (n = 4) as measured by an ELISA assay. Level of NTS presented in (A–C), -7.76 from bregma. Arrows represent dual-labeled cells. CC, central canal. Scale bar, 50 μm applies to (A–C). Scale bar in inset, 25 μm. Data are presented as mean ± SEM, * P < 0.05.

Figure 4.

Pomc ^eGFP cells are responsive to changes in exogenous leptin levels. A–C: Leptin treatment (5 mg/kg, i.p.) in Pomc^eGFP mice increased FOS-IR within GFP-IR (A–B) and non-GFP-IR NTS cells (C) compared to saline treatment (n = 10). Level of NTS presented in A, -7.56 to -7.76 from bregma. D: Representative current clamp recording of a Pomc^eGFP cell. E–F: Bath application of leptin (100–250 nM) increased the membrane potential of 4 out of 10 Pomc^eGFP cells and reduced the membrane potential of 1 cell. G: Representative current clamp recording of a Pomc^eGFP cell in the presence of synaptic blockers (1 μM tetrodotoxin, 3 μM strychnine, 50 μM picrotoxin, 50 μM D-AP5, 10 μM CNQX). H–I: Bath application of leptin (250–500 nM) depolarised and increased the membrane potential of 7 of 30 Pomc^eGFP cells and inhibitory effects were observed in 3 cells. Arrows represent dual-labeled cells. CC, central canal; scale bar A, 50 μm. Data are presented as mean ± SEM. ** P < 0.01, **** P < 0.0001.