Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons

Abstract

Leptin acts in the brain to prevent obesity. The underlying neurocircuitry responsible for this is poorly understood, in part because of incomplete knowledge regarding first-order, leptin-responsive neurons. To address this, we and others have been removing leptin receptors from candidate first-order neurons. While functionally relevant neurons have been identified, the observed effects have been small, suggesting that most first-order neurons remain unidentified. Here we take an alternative approach and test whether first-order neurons are inhibitory (GABAergic, VGAT⁺) or excitatory (glutamatergic, VGLUT2⁺). Remarkably, the vast majority of leptin's antiobesity effects are mediated by GABAergic neurons; glutamatergic neurons play only a minor role. Leptin, working directly on presynaptic GABAergic neurons, many of which appear not to express AgRP, reduces inhibitory tone to postsynaptic POMC neurons. As POMC neurons prevent obesity, their disinhibition by leptin action on presynaptic GABAergic neurons probably mediates, at least in part, leptin's antiobesity effects.

Figure 1. Generation of Vgat-ires-Cre and Vglut2-ires-Cre Mice

(A) Mice expressing Cre recombinase under the control of the endogenous Vgat and Vglut2 genes were generated by inserting an ires-Cre cassette after the Vgat and Vglut2 stop codons, respectively. (B–D) Immunohistochemistry for eGFP expression in Vgat-ires-Cre, lox-GFP mice; brown-staining represents Cre activity. (D) Enlargement of boxed region in (C). (E–G) Immunohistochemistry for eGFP expression in Vglut2-ires-Cre, lox-GFP mice; brown staining represents Cre activity. (G) Enlargement of boxed region in (F). Arc=arcuate, BLA=basolateral amygdala, CeA=central amygdala, CPu=caudate putamen, DMH=dorsomedial hypothalamus, LOT=nucleus of the lateral olfactory tract, PVN=paraventricular nucleus, SCh=suprachiasmatic nucleus, TH=thalamus, VMH=ventromedial hypothalamus, ZI=zona incerta.

Figure 2. Effect of Deleting LEPRs from VGAT⁺ and VGLUT2⁺ Neurons on Body Weight, Body Composition, and Food Intake

(A) Body weight curves of male and female mice with LEPR deleted from VGAT⁺ neurons (open boxes), VGLUT2⁺ neurons (closed triangles) or controls (Lepr^lox/lox, closed circles). Data are presented as mean +/− SEM. Compared to the Lepr^lox/lox controls, all values are significantly different except in week 4 and 7 for the Vglut2-ires-Cre, Lepr^lox/lox females (assessed by one-way ANOVA with Dunnett’s test of all groups compared to Lepr^lox/lox mice ). (B) Fat mass and lean mass of male and female Vgat-ires-Cre, Lepr^lox/lox mice (left) and Vglut2-ires-Cre, Lepr^lox/lox mice (right) at 10 weeks of age were analyzed by EchoMRI. Data are presented as mean +/− SEM.*, p< 0.05; ***, p<0.001; unpaired t-test compared to Lepr^lox/lox controls. (C) Food intake of Vgat-ires-Cre, Lepr^lox/lox mice (left) and Vglut2-ires-Cre, Lepr^lox/lox mice (right). Data are presented as mean +/− SEM.***, p<0.001; unpaired t-test compared to Lepr^lox/lox controls.

Figure 3. GABAergic (VGAT⁺) and Glutamatergic (VGLUT2⁺) Nature of POMC and AgRP Neurons

(A) Colocalization of hrGFP (POMC neurons) and DsRed (GABAergic neurons) in the arcuate in Vgat-ires-Cre, lox-tdTomato, POMC-hrGFP mice. (B) Colocalization of the hrGFP (POMC neurons) and DsRed (glutamatergic VGLUT2⁺ neurons) in the arcuate in Vglut2-ires-Cre, lox-tdTomato, POMC-hrGFP mice. (C) Colocalization of the hrGFP (AgRP/NPY neurons) and DsRed (GABAergic neurons) in the arcuate in Vgat-ires-Cre, lox-tdTomato, NPY-hrGFP mice. Green=anti-hrGFP, Magenta=anti-DsRed, white=hrGFP+DsRed. Scale bar=20 um.

Figure 4. Leptin-induced pSTAT3 Expression in GABAergic and Glutamatergic (VGLUT2⁺) Neurons With and Without LEPRs

(A–F) Double immunohistochemical staining for pSTAT3 and eGFP in the arcuate, DMH, and NTS/DMV from leptin-injected Vgat-ires-Cre, Lepr^+/+, lox-GFP mice (with LEPRs intact, A–C) and Vgat-ires-Cre, Lepr^lox/lox, lox-GFP mice (with LEPRs deleted from GABAergic neurons, D–F). (G–N) Double immunohistochemical staining for pSTAT3 and eGFP in the arcuate, VMH, PMv, and NTS/DMV from leptin-injected Vglut2-ires-Cre, Lepr^+/+, lox-GFP mice (with LEPRs intact, G-J) and Vglut2-ires-Cre, Lepr^lox/lox, lox-GFP mice (with LEPRs deleted from glutamatergic VGLUT2⁺ neurons, K-N). Green=anti-eGFP, Magenta=anti-pSTAT3, white=eGFP+pSTAT3. Scale bar=20 um.

Figure 5. Effects of Leptin Addition on sIPSC Frequency in POMC Neurons

(A) Left–Time course of effects of leptin (100 nM) (blue squares) or of no additions (pink circles) on sIPSC frequency in POMC neurons from control (Lepr^lox/lox) mice. Right - Example traces of sIPSCs recorded from a POMC neuron i) just before and ii) after 25–30 minutes of leptin addition. (B) Effects of 25–30 minutues of leptin treatment on sIPSC frequency, expressed as percent of baseline (prior to leptin addition), in POMC neurons from Lepr^lox/lox (control) mice, Vgat-ires-Cre, Lepr^lox/lox mice, AgRP-ires-Cre, Lepr^lox/lox mice, and POMC-Cre, Lepr^lox/lox mice, and also from Vgat^lox/lox (control) mice and AgRP-ires-Cre, Vgat^lox/lox mice (lacking VGAT in AGRP neurons). Data are presented as mean +/− SEM. **, p<0.01; ***, p<0.001; ****, p<0.0001, paired t-test for effect of leptin versus baseline (see “Supplemental Procedures” for details on how percent baseline was determined).

Figure 6. Effects of Deleting LEPRs Globally (Lepr ^Δ/Δ mice), from GABAergic Neurons, from AgRP neurons or from POMC Neurons on Inhibitory Tone to POMC Neurons

(A) Summary of sIPSC and mIPSC frequency (top left) and amplitude (bottom left) in POMC neurons from Lepr^lox/lox (control) mice, Vgat-ires-Cre, Lepr^lox/lox mice, Lepr^Δ/Δ mice, POMC-Cre, Lepr^lox/lox, and AgRP-ires-Cre, Lepr^lox/lox mice. Data are presented as mean +/− SEM. **, p<0.01; ***, p<0.001; ****, p<0.0001 one-way ANOVA with Dunnett’s test of all groups compared to Lepr^lox/lox mice. (Top right) Example traces of sIPSCs recorded from POMC neurons of a Lepr^lox/lox (control) mouse and a Vgat-ires-Cre, Lepr^lox/lox mouse. (Bottom right) Cumulative probability distribution of mIPSC amplitudes from POMC neurons showing a significant rightward shift in Vgat-ires-Cre, Lepr^lox/lox mice compared to Lepr^lox/lox (control) mice. (B) Summary of membrane potential and firing rate of POMC neurons from Lepr^lox/lox (control) mice and Vgat-ires-Cre, Lepr^lox/lox mice before and after the addition of the GABAA receptor blocker, picrotoxin. Data are presented as mean +/− SEM.

Figure 7. Effects of Fasting on sIPSC Frequency and Amplitude in POMC Neurons

(A) Summary of the effects of fasting on sIPSC frequency (left) and amplitude (right) in POMC neurons of fed Lepr^lox/lox mice, fasted Lepr^lox/lox mice, and fasted+saline- or fasted+leptin-injected Lepr^lox/lox mice. Data are presented as mean +/− SEM. ***, p<0.001 one-way ANOVA with Dunnett’s test of all groups compared to fed Lepr^lox/lox mice. (B) Summary of the effects of fasting on sIPSC frequency (left) and amplitude (right) in POMC neurons of fed and fasted Vgat-ires-Cre, Lepr^lox/lox mice. Data are presented as mean +/− SEM. ***, p<0.001; ****, p<0.0001; t-test compared to fed Vgat-ires-Cre, Lepr^lox/lox mice.