Appropriate inhibition of orexigenic hypothalamic arcuate nucleus neurons independently of leptin receptor/STAT3 signaling

Abstract

Leptin directly suppresses the activity of orexigenic neurons in the hypothalamic arcuate nucleus (ARC). We examined c-Fos-like immunoreactivity (CFLIR) as a marker of ARC neuronal activity in db/db mice devoid of the signaling form of the leptin receptor (LRb) and s/s mice that express LRb(S1138) [which is defective for STAT3 (signal transducer and activator of transcription) signaling]. Both db/db and s/s animals are hyperphagic and obese. This analysis revealed that CFLIR in agouti related peptide-expressing orexigenic ARC neurons is basally elevated in db/db but not s/s mice. Consistent with these observations, electrophysiologic evaluation of a small number of neurons in s/s animals suggested that leptin appropriately suppresses the frequency of IPSCs on ARC proopiomelanocortin (POMC) neurons that are mediated by the release of GABA from orexigenic ARC neurons. CFLIR in POMC neurons of s/s mice was also increased compared with db/db animals. Thus, these data suggest that, although LRb-->STAT3 signaling is crucial for the regulation of feeding, it is not required for the acute or chronic regulation of orexigenic ARC neurons, and the activation of STAT3-mediated transcription by leptin is not required for the appropriate development of leptin responsiveness in these neurons.

Figure 1.

Regulation of CNS CFLIR by altered LRb action. Immunohistochemical detection of CFLIR in fed +/+, db/db, and s/s mice. A, Representative sections of the PVN at bregma level −1.8 mm. B, Representative section of the NTS at bregma level −13.7 mm. C, Representative sections of the ARC at bregma level −3.0 mm, demonstrating increased CFLIR in the ARC of db/db and s/s mice compared with +/+ mice. The ventromedial ARC (right of line) of db/db mice exhibits a dense population of CFLIR-positive neurons that is essentially absent in s/s mice. CFLIR–DAB-positive neurons are visible as brown nuclei. 3V, Third ventricle; AP, area postrema; CC, central canal; ME, median eminence. Photos taken at 10× magnification.

Figure 2.

CFLIR in AgRP neurons of fasted +/+ and ad libitum-fed +/+, db/db, and s/s animals. All mouse groups were bred onto a background expressing LacZ under the AgRP promoter, enabling the identification of AgRP neurons by staining for β-Gal; ad libitum-fed +/+, db/db, and s/s mice and fasted +/+ animals on this background were subjected to IHC analysis. A, Representative images showing immunofluorescent detection of c-Fos (green, top), β-Gal (red, top middle), and merged c-Fos/β-Gal (bottom middle). Bottom, Schematic drawings of AgRP (β-Gal, red × symbols), c-Fos (green circles), and double (AgRP/c-Fos, blue circles) labeled neurons in representative sections (approximately bregma −1.9 mm). Insets show high magnifications of AgRP neurons within the ARC. B, Quantification of double-labeled c-Fos/AgRP neurons from animals treated as in A. Total AgRP neurons were normalized to 100% for the different mouse groups, and double-labeled AgRP neurons are plotted as percentage ± SEM of total AgRP neurons. C, Distribution of double-labeled neurons throughout the ARC. Total c-Fos/AgRP double-labeled neurons per sections are plotted for each genotype/treatment group. Statistical differences between groups were tested by two-way ANOVA (p < 0.0001), and Fisher's PLSD was performed for post hoc analysis (*p < 0.0001 vs fed +/+; ^#p < 0.001 vs fed s/s). Differences in total numbers of AgRP neurons within the groups were tested using ANOVA analysis (p = 0.21) and Fisher's PLSD and showed no significant differences among the groups, although the number of AgRP-positive neurons trended down in db/db mice compared with other groups (p = 0.12 vs +/+). Group sizes were n = 4 (fed +/+, fed db/db, and fed s/s) or n = 3 (fasted +/+). 3V, Third ventricle; ME, median eminence.

Figure 3.

Leptin decreased the frequency of miniature IPSCs onto POMC neurons in s/s mice. A, Ten-second sweeps of raw synaptic data from a single POMC neuron showing baseline IPSCs 45 s before (top trace) and 306 s after (bottom trace) the start of leptin (100 nm) bath application. B, Leptin (100 nm) reversibly decreased the inhibitory synaptic input onto POMC neurons from s/s animals. The shaded region corresponds to time of drug application (n = 2). C, Representative images showing immunohistochemical detection of c-Fos (top, black nuclei) and immunofluorescent detection of β-Gal (middle, β-Gal/POMC, green) (bottom is merge) in db/db and s/s animals with β-Gal expression in POMC neurons (approximately bregma −1.9 mm). White arrows indicate colocalization of CFLIR with β-Gal; similar results were seen in multiple independent animals. Scale bars, 5μm.