Leptin-receptor-expressing neurons in the dorsomedial hypothalamus and median preoptic area regulate sympathetic brown adipose tissue circuits - PubMed (original) (raw)

Leptin-receptor-expressing neurons in the dorsomedial hypothalamus and median preoptic area regulate sympathetic brown adipose tissue circuits

Yan Zhang et al. J Neurosci. 2011.

Abstract

Brown adipose tissue (BAT) thermogenesis is critical to maintain homoeothermia and is centrally controlled via sympathetic outputs. Body temperature and BAT activity also impact energy expenditure, and obesity is commonly associated with decreased BAT capacity and sympathetic tone. Severely obese mice that lack leptin or its receptor (LepRb) show decreased BAT capacity, sympathetic tone, and body temperature and thus are unable to adapt to acute cold exposure (Trayhurn et al., 1976). LepRb-expressing neurons are found in several hypothalamic sites, including the dorsomedial hypothalamus (DMH) and median preoptic area (mPOA), both critical sites to regulate sympathetic, thermoregulatory BAT circuits. Specifically, a subpopulation in the DMH/dorsal hypothalamic area (DHA) is stimulated by fever-inducing endotoxins or cold exposure (Dimicco and Zaretsky, 2007; Morrison et al., 2008). Using the retrograde, transsynaptic tracer pseudorabies virus (PRV) injected into the BAT of mice, we identified PRV-labeled LepRb neurons in the DMH/DHA and mPOA (and other sites), thus indicating their involvement in the regulation of sympathetic BAT circuits. Indeed, acute cold exposure induced c-Fos (as a surrogate for neuronal activity) in DMH/DHA LepRb neurons, and a large number of mPOA LepRb neurons project to the DMH/DHA. Furthermore, DMH/DHA LepRb neurons (and a subpopulation of LepRb mPOA neurons) project and synaptically couple to rostral raphe pallidus neurons, consistent with the current understanding of BAT thermoregulatory circuits from the DMH/DHA and mPOA (Dimicco and Zaretsky, 2007; Morrison et al., 2008). Thus, these data present strong evidence that LepRb neurons in the DMH/DHA and mPOA mediate thermoregulatory leptin action.

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Figures

Figure 1.

Figure 1.

Neuroanatomical localization of LepRb neurons in the DMH. A, B, Immunohistochemical staining of a LepRbEGFP mouse brain for Nissl (top), NeuN (middle), and counterstained with LepRbEGFP (bottom). In the overview (A), the outline of the DMH with regard to the third ventricle (3V), mt, and fx are shown. In the high magnification (B), the compact DMH is seen by the more cell-dense area in the DMH, and dDMH and vDMH are located dorsal and ventral to the cDMH, respectively. The DHA is defined as the area between the mt and dDMH. C, Rostral to caudal extend of LepRb neurons in the DMH in relation to the neuronal marker NeuN and mt as a landmark.

Figure 2.

Figure 2.

Neuroanatomical location of LepRb neurons in the mPOA. A, B and C, D show two mPOA levels in a LepRbEGFP mouse stained for LepRbEGFP (B, D) and the neuronal marker NeuN (A–D). LepRb neurons are found in the MPA and MnPO (collectively referred to as mPOA).

Figure 3.

Figure 3.

LepRbEGFP mice treated with PBS (A) or leptin (1 mg/kg; B) and stained for pSTAT3 (nuclear black stain) and EGFP (green) to demonstrate leptin-induced pSTAT3 in LepRbEGFP neurons (n = 3). C, Schematic drawing of the area in A and B.

Figure 4.

Figure 4.

Representative images from LepRbEGFP mice 96 h after PRV infection of the BAT. Transsynaptically and retrogradely PRV (red stain) traced LepRbEGFP (greed stain) neurons are found in the DMH/DHA (A), mPOA (B), RCh (C), NTS (D), and EW (E). Other PRV- and LepRb-positive sites, such as the lateral hypothalamic area (LHA, F), did not show colocalized neurons.

Figure 5.

Figure 5.

Quantification of PRV/_LepRbEGFP_-positive neurons. Percentage of double-labeled neurons compared with total PRV-labeled neurons in different brain sites (n = 3).

Figure 6.

Figure 6.

A, Immunohistochemistry of c-Fos induction (red) in LepRbEGFP neurons (green) after 3 h acute cold exposure (n = 6) compared with room temperature (RT) conditions (n = 5). B, Percentage of colocalized c-Fos/LepRbEGFP neurons relative to total LepRbEGFP neurons in the DMH/DHA at room temperature versus 3 h acute cold exposure. C, Total number of LepRbEGFP neurons in the DMH/DHA of mice kept at room temperature or after 3 h cold exposure. *p < 0.00000001, t test. 3V, Third ventricle.

Figure 7.

Figure 7.

Representative images of a LepRbcre/cre mouse with DMH-specific injection of cre-inducible Ad-iZ/EGFPf (n = 4). Immunohistochemical detection of EGFPf shows projections into the mPOA (A), PVN (B), DMH (C), and rRPa (D).

Figure 8.

Figure 8.

Stereotaxic injection of the retrograde tracer FG into the RMR (A, n = 4) and immunohistochemical detection of retrogradely labeled FG (green) in LepRb neurons (leptin-induced pSTAT3, red) in the DMH/DHA (B) and mPOA (C). Schematic drawings of double-labeled FG/pSTAT3 neurons (red dots) in contrast to non-FG-labeled pSTAT3 neurons (green dots) in the DMH/DHA (B′) and mPOA (C′). 3V, Third ventricle.

Figure 9.

Figure 9.

Stereotaxic injection of the retrograde tracer FG into the PVN (A, n = 2) and immunohistochemical detection of retrogradely labeled FG (green) in LepRb neurons (leptin-induced pSTAT3, red) in the DMH/DHA (B).

Figure 10.

Figure 10.

LepRb neurons are synaptically coupled with neurons in the rRPa. In LepRbEGFP reporter mice, _LepRbEGFP_-positive fibers (green) are seen in close proximity to neurons in the rRPa (red) (B, n = 10). The same section is shown as an overview with NeuN stain in A. Transsynaptically labeled WGA-positive neurons are found in the rRPa of LepRbWGA mice (D, n = 5), indicating synaptic coupling of LepRb neurons with rRPa neurons. An overview of the same sections is shown in C. 4V, Fourth ventricle; py, pyramidal tract.

Figure 11.

Figure 11.

Stereotaxic injection of the retrograde tracer FG into the DMH/DHA (A, n = 3) and IHC for leptin-induced pSTAT3 (B) to demonstrate the location of the FG injection relative to LepRb neurons in the DMH/DHA. Immunohistochemical detection of retrogradely labeled FG (green) in LepRb neurons (leptin-induced pSTAT3, red) in the mPOA (C) and schematic drawing of double-labeled FG/pSTAT3 neurons (red dots) in contrast to non-FG-labeled pSTAT3 neurons (green dots) in the mPOA (D).

Figure 12.

Figure 12.

Schematic drawing of identified LepRb connections involved in thermoregulatory circuits.

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