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1.
Figure 1

Figure 1. LepRbcre-dependent transgenic and adenoviral tracers to study LepRb-expressing cells in the LHA. From: Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons.

(A) Schematic diagram showing mechanisms of cre-mediated tracer (EGFP, WGA, or EGFPf) expression in LepRb-expressing cells of LepRbEGFP mice and LepRbEGFP/WGA mice and also upon injection of Ad-iZ/EGFPf and Ad-iN/WED adenoviruses. (B, C) Immunofluorescent detection of EGFP (green) and (B) MCH (red) or (C) orexin (red) in the LHA of LepRbEGFP mice. Insets: represent digitally zoomed images of labeled LHA neurons. Scale bars = 200µm, 3V = third ventricle; f = fornix.

Gwendolyn W. Louis, et al. J Neurosci. ;30(34):11278-11287.
2.
Figure 7

Figure 7. Changes in gene expression after systemic and local leptin treatment in Lepob/ob (ob/ob) mice. From: Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons.

Changes in gene expression in microdissected tissue of ARC or LHA from Lepob/ob mice following 26h (A) systemic (IP, 5 mg/kg) or (B) intra-LHA (0.25 ng) treatment with PBS (black bars) or leptin (gray bars). Expression data are plotted relative to Gapdh expression (calculated by 2−ΔΔCt method) and normalized to control (PBS-treated) levels and contralateral sides (for intra-LHA group) ± SEM. *p<0.05, **p<0.01 relative to PBS.

Gwendolyn W. Louis, et al. J Neurosci. ;30(34):11278-11287.
3.
Figure 6

Figure 6. Local tracing of LepRb neurons and their synaptic targets in the LHA after Ad-iN/WED injection to the LHA of LepRbcre mice. From: Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons.

Immunofluorescent detection of (A&E) WGA (green) and (A) MCH (red) or (E) OX (red) in the LHA after stereotaxic injection of Ad-iN/WED to the LHA. Panels B–D and F–H show single channel and merged enlarged images of the boxed regions from A and E, respectively. Insets: digitally magnified view of labeled neurons. * indicates injection site; arrows = colabeled cells. Scale bars = 200µm, 3V = third ventricle; f = fornix.

Gwendolyn W. Louis, et al. J Neurosci. ;30(34):11278-11287.
4.
Figure 4

Figure 4. Ad-iZ/EGFPf injection to the LHA of LepRbcre mice provides evidence for intra-LHA as well as intra-VTA synapses on projections from LHA LepRb neurons. From: Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons.

Immunofluorescent detection of EGFPf in the (A–C) LHA and (D–F) in the VTA. Panels B,C and E,F represent digitally zoomed images of synaptic boutons in panels A and D, respectively. Confocal images of (G) EGFP/LepRb (green) and (H) OX (red) neurons within the LHA. Panels I–L represent merged images; J–L are digitally magnified views of synaptic boutons from the boxed region in panel I. Scale bars A&D=200µm, G–I= 50µm. 3V = third ventricle; f = fornix; ip = intrapenduncular nucleus; ml = medial lemiscus

Gwendolyn W. Louis, et al. J Neurosci. ;30(34):11278-11287.
5.
Figure 2

Figure 2. LepRbEGFP/WGA mice reveal LepRb-expressing cells and their projection targets in the LHA. From: Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons.

(A) Immunofluorescent detection of WGA (red) and GFP/LepRb (green) in the LHA of LepRbEGFP/WGA mice. Immunofluorescent detection of (E, I) WGA (green) and (E) MCH (red) and (I) OX (red) in the LHA of LepRbEGFP/WGA mice. Panels B–D, F–H, and J–L show enlarged images of the boxed regions of panels A, E, and I, respectively. Panels B, F, and J show red channel only, C, G, and K show green channel only, and D, H, and L show merged images. Insets: higher magnification view of boxed regions. Yellow arrows= representative colabeled cells; white arrowheads = single labeled cells. Scale bars = 200µm, 3V = third ventricle; f = fornix.

Gwendolyn W. Louis, et al. J Neurosci. ;30(34):11278-11287.
6.
Figure 5

Figure 5. Modest accumulation of WGA in VTA relative to LHA neurons following Ad-iN/WED injection to the LHA of LepRbcre mice. From: Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons.

(A) Immunofluorescent detection of a robust population of WGA-IR neurons (green) in the LHA of a representative intra-LHA Ad-iN/WED-injected LepRbcre mouse. (B) Enlarged image of the boxed region in A. (C) Detection of WGA (green) and a robust population of TH-IR neurons (DA; red) in the VTA. (D–F) Show single channel and merged enlarged images of the boxed region from C. * indicates injection site; arrows = representative colabeled cells. Scale bars = 200µm, unless otherwise noted. 3V = third ventricle; f = fornix; ip = intrapenduncular nucleus.

Gwendolyn W. Louis, et al. J Neurosci. ;30(34):11278-11287.
7.
Figure 3

Figure 3. Retrograde tracing from the LHA OX field in LepRbEGFP mice to determine potential presynaptic LepRb populations. From: Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons.

The retrograde tracer, fluorogold (FG), was injected into the dorsal perifornical region of the LHA of LepRbEGFP mice; after perfusion, the brains were processed for the immunofluorescent detection of GFP/LepRb (green) and FG (red). (A) View of the LHA injection site in a representative animal. (B) ARC, (C, D) VTA and (E, F) lateral POA. Panels B,D,F are magnified images of boxed areas in A,C,E, respectively. Representative images from one of two animals with injection site restricted to the dorsal periformnical area are shown. Arrows= representative colabeled cells. Scale bars = 200µm, unless otherwise noted. 3V = third ventricle; f = fornix; ip = intrapenduncular nucleus; ml = medial lemiscus; ac = anterior commissure.

Gwendolyn W. Louis, et al. J Neurosci. ;30(34):11278-11287.

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