Neurochemical characterization of body weight-regulating leptin receptor neurons in the nucleus of the solitary tract

Abstract

The action of peripherally released leptin at long-form leptin receptors (LepRb) within the brain represents a fundamental axis in the regulation of energy homeostasis and body weight. Efforts to delineate the neuronal mediators of leptin action have recently focused on extrahypothalamic populations and have revealed that leptin action within the nucleus of the solitary tract (NTS) is critical for normal appetite and body weight regulation. To elucidate the neuronal circuits that mediate leptin action within the NTS, we employed multiple transgenic reporter lines to characterize the neurochemical identity of LepRb-expressing NTS neurons. LepRb expression was not detected in energy balance-associated NTS neurons that express cocaine- and amphetamine-regulated transcript, brain-derived neurotrophic factor, neuropeptide Y, nesfatin, catecholamines, γ-aminobutyric acid, prolactin-releasing peptide, or nitric oxide synthase. The population of LepRb-expressing NTS neurons was comprised of subpopulations marked by a proopiomelanocortin-enhanced green fluorescent protein (EGFP) transgene and distinct populations that express proglucagon and/or cholecystokinin. The significance of leptin action on these three populations of NTS neurons was assessed in leptin-deficient Ob/Ob mice, revealing increased NTS proglucagon and cholecystokinin, but not proopiomelanocortin, expression. These data provide new insight into the appetitive brainstem circuits engaged by leptin.

Fig. 1.

Neuroanatomical distribution and validation of transgenically labeled LepRb-expressing neurons of the NTS. LepRb-expressing neurons in LepRb-Ires-Cre::tdTOM and LepRb-Cre::EYFP mice were found across the rostral-caudal extent of the NTS with the preponderance being localized to the NTS at the level of the area postrema. The validity of reporter expression in each line was confirmed via leptin-induced pSTAT3-IR and colocalization with tdTOM- or EYFP-expressing neurons. Each dot () represents five reporter-labeled neurons negative for pSTAT3 and each star (★) five reporter-labeled neurons positive for pSTAT3. AP, Area postrema; cc, central canal; 4v, fourth ventricle.

Fig. 2.

Neurochemical profiling of LepRb-expressing cells of the caudal NTS. A–F, Double-immunofluorescent analysis of LepRb-expressing neurons in compound LepRb-Ires-Cre::tdTOM/POMC-EGFP and LepRb-Ires-Cre::tdTOM/NPY-GFP mice revealed colocalization of LepRb-expressing neurons (red) with POMC-expressing (green; A) but not NPY-expressing (green; B) neurons of the NTS. Immunohistological analysis in LepRb-Cre::EYFP mice also demonstrated these cells to be negative for TH (C), nesfatin (D), ChAT (E), nNOS (F). G–L, Dual-immunohistological and in situ hybridization analysis demonstrated the absence of LepRb coexpression (brown cytoplasmic stain) with PrRP (G), GAD67 (H), BDNF (I), or CART (J) mRNA (black grains). However, LepRb neurons were found to express PPG/GLP1 (K) and CCK (L) mRNA. M–O, Population analysis of NTS LepRb-expressing neurons demonstrated that POMC-EGFP neurons did not express PPG/GLP1 (M) or CCK (N) but that a proportion of PPG/GLP1-YFP NTS neurons also exhibited CCK expression (O). Arrows represent single-labeled cells and arrowheads colocalized cells. cc, Central canal. Scale bar in A applies to A–I and represents 50 μm. Scale bar in A inset applies to insets in K, L, and O and represents 25 μm.

Fig. 3.

The effect of leptin deficiency on NTS LepRb neuron-associated neuropeptide expression. Quantitative radioactive in situ hybridization autoradiographical analysis of POMC (A–C), PPG/GLP1 (D–F), and CCK (G–I) mRNA expression in leptin-deficient Ob/Ob mice. Loss of leptin tone had no effect on NTS POMC expression but resulted in a significant up-regulation of PPG/GLP1 and CCK expression. C, F, and I, Data are presented as mean ± sem, Student's t test. *, P < 0.05.