Ventral tegmental area leptin receptor neurons specifically project to and regulate cocaine- and amphetamine-regulated transcript neurons of the extended central amygdala - PubMed (original) (raw)

Comparative Study

Ventral tegmental area leptin receptor neurons specifically project to and regulate cocaine- and amphetamine-regulated transcript neurons of the extended central amygdala

Rebecca L Leshan et al. J Neurosci. 2010.

Abstract

Leptin acts via its receptor (LepRb) to regulate neural circuits in concert with body energy stores. In addition to acting on a number of hypothalamic structures, leptin modulates the mesolimbic dopamine (DA) system. To determine the sites at which LepRb neurons might directly influence the mesolimbic DA system, we examined the distribution of LepRb neurons and their projections within mesolimbic brain regions. Although the ventral tegmental area (VTA) contains DA LepRb neurons, LepRb neurons are absent from the amygdala and striatum. Also, LepRb-EGFPf mice (which label projections from LepRb neurons throughout the brain) reveal that few LepRb neurons project to the nucleus accumbens (NAc). In contrast, the central amygdala (CeA) and its rostral extension receive copious projections from LepRb neurons. Indeed, LepRb-specific anterograde tracing demonstrates (and retrograde tracing confirms) that VTA LepRb neurons project to the extended CeA (extCeA) but not the NAc. Consistently, leptin promotes cAMP response element-binding protein phosphorylation in the extCeA, but not NAc, of leptin-deficient animals. Furthermore, transgenic mice expressing the trans-synaptic tracer wheat germ agglutinin in LepRb neurons reveal the innervation of CeA cocaine- and amphetamine-regulated transcript (CART) neurons by LepRb neurons, and leptin suppresses the increased CeA CART expression of leptin-deficient animals. Thus, LepRb VTA neurons represent a subclass of VTA DA neurons that specifically innervates and controls the extCeA; we hypothesize that these neurons primarily modulate CeA-directed behaviors.

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Figures

Figure 1.

Figure 1.

Mouse models and the visualization of midbrain LepRb neurons. A, Schematic of methods for expression of EGFP or EGFPf in LepRb neurons. Combining Leprcre with Rosa26-EGFP or Rosa26-EGFPf alleles results in the stable expression of EGFP or EGFPf in LepRb neurons in LepRbEGFP and LepRbEGFPf mice, respectively (top). Additionally, injection into Leprcre mice of the adenoviral Ad-iZ/EGFPf promotes cre-mediated EGFPf expression in LepRb neurons surrounding in the injection site (bottom). 3′UTR, 3′ untranslated region. B, D, F, LepRb-expressing neurons revealed by EGPF immunoreactivity through the rostrocaudal extent of the midbrain of LepRbEGFP animals. C, E, G, Colocalization of EGFP (green) and TH (red) immunoreactivity through the rostrocaudal extent of the midbrain of LepRbEGFP mice. Insets show digital zooms of the boxed areas; arrows demonstrate examples of colocalized neurons. Red asterisks indicate the medial lemniscus; × indicates the ventral tegmental decussation. Scale bar, 200 μm. Aq, Central aqueduct; IP, interpeduncular nucleus; IF, interfascicular nucleus.

Figure 2.

Figure 2.

Detection of LepRb neurons and projections throughout the mesolimbic DA system in LepRbEGFP and LepRbEGFPf mice. A–H, EGFP immunoreactivity in the midbrain (A, B), hypothalamus and amygdala (C, D), rostral hypothalamus and IPAC (E, F), and striatum and BNST (G, H) of LepRbEGFP (left) and LepRbEGFPf (right) mice is shown. Arrows in C and D indicate the CeA; arrows in E and F indicate the IPAC. The dashed × indicates the ventral tegmental decussation. Scale bars, 200 μm. ml, Medial lemniscus; IP, interpeduncular nucleus; opt, optic tract; 3v, third ventricle; f, fornix; LV, lateral ventricle; ac, anterior commissure.

Figure 3.

Figure 3.

CREB phosphorylation in the midbrain, amygdala, and NAc of leptin-treated Lepob/ob mice. Leptin-deficient Lepob/ob (ob/ob) mice were treated with leptin (5 mg/kg, i.p., 2 h) and perfused for the immunohistochemical detection of pCREB immunoreactivity. A–H, Representative images of pCREB immunoreactivity in VTA (A, E), amygdala (B, F), IPAC (C, G), and NAc (D, H) of vehicle (top) and leptin-treated (bottom) animals. Circles denote regions analyzed for staining intensity, which is plotted as mean ± SEM in I. n = 6 for leptin treated and n = 5 for PBS treated. *p < 0.05. Scale bars, 100 μm.

Figure 4.

Figure 4.

Representative Ad-iZ/EGFPf-mediated tracing of projections primarily from VTA LepRb neurons in Leprcre mice. A, B, Schematic (A) and EGFP immunoreactivity (B) of the VTA injection site in a representative case. C, The appearance of rostral projections (red) in this animal is superimposed on sections from the atlas of Paxinos and Franklin (2001). D–G, EGFP immunoreactivity in various regions to which VTA LepRb neurons sent detectable projections. Insets represent digital zooms of boxed regions. The arrow in G indicates the small amount of NAc EGFP immunoreactivity observed in this and similar cases. Red asterisks indicate the medial lemniscus. Scale bars: B, D, 200 μm; E–G, 100 μm. IP, Interpeduncular nucleus; acp, anterior commissure; ac, anterior commissure; LV, lateral ventricle.

Figure 5.

Figure 5.

Representative Ad-iZ/EGFPf-mediated tracing of projections from VTA and midline midbrain LepRb neurons in Leprcre mice. A, B, Schematic (A) and EGFP immunoreactivity (B) of the midbrain injection site in a representative case. C, The appearance of rostral projections (red) in this animal is superimposed on sections from the atlas of Paxinos and Franklin (2001). D–G, EGFP immunoreactivity in various regions to which midbrain LepRb neurons sent detectable projections. Insets represent digital zooms of boxed regions. Red asterisks indicate the medial lemniscus. Scale bars, 200 μm. IP, Interpeduncular nucleus; acp, anterior commissure; ac, anterior commissure; LV, lateral ventricle; opt, optic tract.

Figure 6.

Figure 6.

Retrograde tracing from CeA labels VTA LepRb neurons. The retrograde tracer FG was stereotaxically injected into the CeA of LepRbEGFP animals to determine the potential projection of VTA LeRb neurons to the CeA by colocalization of FG and EGFP immunoreactivity. A, B, Schematic diagram (A) and fluorescent image (B; red, FG; green, EGFP) of the CeA injection site in a representative animal. C, D, Distribution of FG- and EGFP-IR neurons at two different levels of the VTA. Images below are digital zooms of the boxed areas showing (left to right) merged images, FG immunoreactivity, and EGFP immunoreactivity. Arrows indicate colocalized neurons. Red asterisks indicates the BLA. Scale bars: B, C, 200 μm; insets, 25 μm. IP, Interpeduncular nucleus; ml, medial lemniscus.

Figure 7.

Figure 7.

Retrograde tracing from IPAC labels VTA LepRb neurons. The retrograde tracer FG was stereotaxically injected into the IPAC of LepRbEGFP animals to determine the potential projection of VTA LeRb neurons to the IPAC by colocalization of FG and EGFP immunoreactivity. A, B, Schematic diagram (A) and fluorescent image (B; red, FG; green, EGFP) of the IPAC injection site in a representative animal. C, Distribution of FG- and EGFP-IR neurons in the VTA. Images below are digital zooms of the boxed areas showing (top to bottom) merged images, FG immunoreactivity, and EGFP immunoreactivity. Arrows indicate colocalized neurons. Red asterisks indicate the BLA. Scale bars: B, C, 200 μm; insets, 20 μm. IP, Interpeduncular nucleus; ml, medial lemniscus; 3v, third ventricle.

Figure 8.

Figure 8.

Retrograde tracing from NAc labels midline midbrain but not VTA LepRb neurons. The retrograde tracer FG was stereotaxically injected into the NAc of LepRbEGFP animals to determine the potential projection of VTA LeRb neurons to the NAc by colocalization of FG and EGFP immunoreactivity. A, B, Schematic diagram (A) and fluorescent image (B; red, FG; green, EGFP) of the NAc injection site in a representative animal. C, D, Distribution of FG- and EGFP-IR neurons in the VTA. Images below are digital zooms of the boxed areas showing (top to bottom) merged images, FG immunoreactivity, and EGFP immunoreactivity. Arrows indicate colocalized neurons. Red asterisks indicate the anterior commissure. Scale bars: B, C, 200 μm; insets, 25 μm. IP, Interpeduncular nucleus; ml, medial lemniscus; LV, lateral ventricle.

Figure 9.

Figure 9.

Identification of CART-expressing CeA neurons as targets of leptin action. A, Schematic diagram showing the generation of LepRb-WGA mice. Leprcre mice were crossed with iZ/WAP transgenic mice to mediate the expression of the trans-synaptic tracer WGA in LepRb neurons. IRES, Internal ribosome entry site; AP, alkaline phosphatase; pA, polyadenylation site. B, WGA immunoreactivity in the hypothalamus and amygdala of a LepRb-WGA mouse. opt, optic tract. Inset, Higher-magnification image showing WGA immunoreactivity in the CeA. Scale bars, 200 μm. C–E, WGA-IR (C, green), CART-IR (D, red), and merged (E) confocal images from the CeA of a LepRb-WGA mouse. Arrows indicate colocalized neurons. Scale bars are as indicated. F, Wild-type (WT) and leptin-deficient Lepob/ob (ob/ob) mice were treated with leptin (5 mg/kg, i.p.) or vehicle 12 h for 24 h before dissection and mRNA extraction from the CeA. Expression of Cart mRNA was quantified by quantitative PCR and is plotted as mean ± SEM. n = 9–10 per group; *p < 0.05, compared with WT by ANOVA.

Figure 10.

Figure 10.

LepRb neurons originating in the midbrain have specific and circumscribed targets in striatal projection regions. Model describing projection patterns of LepRb neurons that originate in the VTA, which are primarily DAergic and project extensively to the CeA and IPAC; within the CeA, these projections innervate and regulate CART neurons. LepRb neurons that originate in the midline RLi project primarily to the IPAC but also send some projections to the NAc.

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