Figure 1.
Leptin activates STAT3 phosphorylation in hypothalamic nuclei of C57BL/6J mice.
Shown are light microscopy (LM) images of phospho-STAT3 immunoreactivity (IR) (DAB) in coronal brain sections of the mediobasal hypothalamus from 8-12 weeks old wild type C57BL/6J male mice. Animals were given vehicle or leptin (5 mg/kg, ip) and sacrificed after 30 minutes. Weak pSTAT3 IR is detected in the arcuate nucleus of the vehicle treated animals. In contrast, robust pSTAT3 IR is found in the LHA, VMH, DMH, Arc and PMN of the leptin-treated mouse. Three matched rostral-to-caudal sections are shown for both treatment groups. Boxes 1-4 are magnified in Figure 2. LHA: lateral hypothalamic area; VMH: ventro-medial hypothalamic nucleus; DMH: dorso-medial hypothalamic nucleus; Arc: arcuate hypothalamic nucleus; PMN: premammillary hypothalamic nucleus; fx: fornix, PH: posterior hypothalamic region; 3v: 3rd ventricle.
Figure 2.
Leptin activates STAT3 phosphorylation in neuronal processes within hypothalamic nuclei of C57BL/6J mice.
High-magnification LM microphotographs of numbered boxes in Figure 1. Many pSTAT3 IR neuronal fibers are observed in all nuclei. LHA: lateral hypothalamic area; VMH: ventro-medial hypothalamic nucleus; DMH: dorso-medial hypothalamic nucleus; Arc: arcuate hypothalamic nucleus; PMN: premammillary hypothalamic nucleus; fx: fornix, 3v: 3rd ventricle.
Figure 3.
Leptin activates STAT3 phosphorylation in soma and processes of hypothalamic POMC neurons of mice.
Top Row: Pomc-EGFP mice (6-10 wks of age) were given leptin (5 mg/kg, ip, 30 min). Shown is confocal laser scanning microscopy (CLSM) of fluorescent pSTAT3 IR (red) and EGFP-epifluorescence (green) in a brain section of the arcuate nucleus of the hypothalamus. pSTAT3 IR is observed both in the nucleus and cytoplasm, as well as in several fibers (arrows) of POMC neurons. Bottom Row: High magnification microphotographs of boxes in top panels. All images are single confocal planes.
Figure 4.
Localization of HA-tagged LepRb at the plasma membrane and cytoplasm of hypothalamic POMC neurons.
A. CLSM of brain sections from a Pomc-cre;HA-Leprb flox;EGFP flox mouse. Shown is the soma of a hypothalamic POMC neuron expressing HA-LepRb (EGFP IR (red); HA IR (green); Nuclear DAPI (blue)). Within the cytoplasm, HA-LepRb is expressed in a vesicular/punctate pattern. Right: Enlargement of box area in A, depicting plasma membrane localization of HA-LepRb (arrows). Shown are single plane sections (0.42 µm). B. Left: CLSM was used to generate a collapsed Z-stack of the soma of a hypothalamic POMC neuron (EGFP IR (red)) expressing HA-LepRb (HA IR (green)). The Z-stack is ~16 µm deep comprising of 39 confocal sections. Right: The soma of the neuron is reconstructed in 3-dimensions using all 39 sections and the Reconstruct computer software. A non-transparent surface representation of the cell is shown in red (insert). A grey semi-transparent surface representation is also depicted. Blue is the nucleus (non-transparent). A number of vesicular-like structures that express HA-LepRb are shown in green. In addition, peri-nuclear localization of HA-LepRb in the presumed Golgi/trans-Golgi networks is represented in orange.
Figure 5.
Localization of HA-tagged LepRb in proximal neuronal fibers of hypothalamic POMC neurons.
CLSM was used to generate a collapsed Z-stack of a POMC neuron from a Pomc-cre;HA-Leprb flox;EGFP flox mouse. The soma and several proximal neuronal fibers can be seen. HA-LepRb is observed in a punctate pattern in three primary and one secondary fiber processes. Due to high background in the Z-stack and to more clearly identify the fibers, areas that not are associated with the POMC neuron have been blacked out (*). Bottom: Enlargement of boxes showing punctuate localization of HA-LepRb IR along a proximal POMC fiber.
Figure 6.
HA-LepRb is localized to dendrites, but not axons, of POMC neurons.
A. Middle Image (*): CLSM was applied to depict a POMC neuron with a >200 µm long dendrite (4 arrow heads) in a brain section from a Pomc-cre;HA-Leprb flox mouse. POMC IR is shown in red and HA (LepRb) IR in green. To present the entire length of the dendrite, the image was merged from three different focal planes. Left Column: The POMC soma expresses both HA-LepRb and POMC polypeptides. None of the POMC axonal fibers (red arrows) in the field exhibit HA IR. Right Image. Magnification of box area depicting a fiber segment containing punctate HA-LepRb expression. POMC IR is not detectable in the fiber (dendrite). Shown are single confocal planes. B. Depicted are two POMC somata co-expressing β-endorphin IR (red) and HA-LepRb (HA IR (green)) in a brain section from a Pomc-cre;HA-Leprb flox mouse. Two nearby POMC IR-axonal fibers (arrows) do not express HA-LepRb. Nuclear DAPI is colored blue. These are single confocal planes.
Figure 7.
Leptin does not activate STAT3 phosphorylation in α-MSH-containing axonal fibers of POMC neurons.
CLSM showing pSTAT3 IR (red) and α-MSH IR (green) and DAPI (blue) in the arcuate nucleus of a leptin-treated (5 mg/kg; 30 min) Leprbdb/db;Pomc-cre;HA-Leprb flox mouse (8 weeks old). Top: Shown are single confocal planes. Examples of two POMC somata with nuclear pSTAT3 and cytoplasmic α-MSH are indicated with arrows in merged image. 3v; 3rd ventricle. Bottom: Magnification of box area. Many α-MSH IR axonal fibers with boutons are found (single arrows). These do not co-express pSTAT3. In contrast, pSTAT3 IR fibers (double arrows) do not exhibit α-MSH IR and do not show presence of axonal boutons.
Figure 8.
Activation of STAT3 phosphorylation by leptin in dendrites of hypothalamic POMC neurons by immuno-electron microscopy (EM).
Immuno-EM for pSTAT3 (DAB) in a leptin-treated (4 mg/kg ip; 10 min) Leprdb/db;Pomc-cre;HA-LepRb flox mouse. Left image: EM scope magnification is 440x showing an overview of the medial arcuate hypothalamic region including the ependymal cells lining the 3rd ventricle (3v). A pSTAT3 IR neuronal nucleus is marked with a black arrow (Nu) and a pSTAT3-negative nucleus is labeled with a white arrow (Nu). Top Right: pSTAT3 IR dendrite in the horizontal plane (EM @ 1,200x). Bottom Right: Cross-section of pSTAT3 IR dendrite (EM @ 30,000x). A mitochondrion (M) and arrays of microtubules (MT) are seen.
Figure 9.
Activation of STAT3 phosphorylation by leptin in dendrites of hypothalamic AgRP neurons by immuno-electron microscopy (EM).
Immuno-EM for pSTAT3 (DAB) in a leptin-treated (4 mg/kg ip; 10 min) Leprdb/db;Agrp-ires-cre;HA-LepRb flox mouse. Left Top: Overview of medial-arcuate region. Top Right: Magnification of box from left. pSTAT3 IR in a neuronal nucleus (“Nu” in black text) and a non-pSTAT3 IR nucleus is labeled with “Nu” (white text). Bottom Left: Cross-section of a pSTAT3 IR dendrite (EM @ 30,000x). Bottom Right: A pSTAT3 IR dendrite in the horizontal plane (EM @ 1,200x). bv: blood vessel.
Figure 10.
Leptin activates STAT3 signaling in proximity to post-synaptic structures on dendritic shafts from POMC neurons.
Top Left: Immuno-EM for pSTAT3 (DAB) from a leptin-treated (4 mg/kg ip; 10 min) Leprdb/db;POMC-cre;HA-LepRb flox mouse. Arrow indicates an asymmetrical synapse on a cross section of a pSTAT3 IR dendritic shaft (D). The presynaptic nerve terminal is labeled (NT). A non-pSTAT3 IR dendrite is also marked (D*). Top Right: Example of a pSTAT3 IR dendritic shaft with a symmetrical synapse (double arrows). Bottom Left: Immuno-Gold EM for pSTAT3 from a leptin-treated (4 mg/kg ip; 10 min) Leprdb/db;POMC-cre;HA-LepRb flox mouse. Arrow indicates PSD of an asymmetrical synapse. Bottom Right: Immuno-EM for GFP (DAB) from a Pomc-cre;EGFP flox mouse. Arrow indicates an asymmetrical synapse on a cross section of a GFP IR (POMC) dendritic shaft. PSD: post-synaptic density.
Figure 11.
Leptin activates STAT3 signaling in proximity to post-synaptic structures on dendritic shafts from AgRP neurons.
Left Side: Immuno-EM for pSTAT3 (DAB) from a leptin-treated (4 mg/kg ip; 10 min) Leprdb/db;AgRP-cre;HA-LepRb flox mouse. The dendrite (D) is marked by black stippled line. Bottom: Magnification of symmetrical synaptic structure from stippled box above. Double arrows indicate the PSD. Right Side: Immuno-EM for pSTAT3 (DAB) in cross section of dendritic shaft from a leptin-treated (4 mg/kg ip; 10 min) Leprdb/db;AgRP-cre;HA-LepRb flox mouse. The dendrite (D) is marked by a black stippled line. Bottom: Magnification of the asymmetrical synapse from above. Single black arrow indicates the PSD. D: dendrite (pSTAT3 IR positive); D*: dendrite (pSTAT3 IR negative); M: mitochondrion; NT: presynaptic nerve terminal.
Figure 12.
3D-reconstruction of a leptin-responsive hypothalamic POMC dendritic segment.
A. Immuno-EM for pSTAT3 (DAB) in a leptin-treated Leprdb/db;Pomc-cre;HA-LepRb flox mouse. Cross-section of a pSTAT3 IR dendritic shaft with a single mitochondrion are shown. Two non-pSTAT3 IR dendrites also marked (D*). B. Serial-EM 3D-reconstruction of the POMC dendritic shaft of the pSTAT3 IR dendrite in Figure A. Top: Shown is a partially transparent presentation (mitochondrion in green and the dendritic surface in gray). Middle: Non-transparent surface representation of same POMC dendritic shaft segment (after rotation). Bottom: Shown is a partially transparent representation with shaft synapses depicted in red (N=15). Total length of segment is ~8.0 µm (reconstructed from 160 sections - each ~50 nm thick). D: dendrite; M: mitochondrion.
Figure 13.
3D-reconstruction of a leptin-responsive hypothalamic AgRP dendritic segment.
A. Immuno-EM for pSTAT3 (DAB) in a leptin-treated Leprdb/db;AgRP-ires-cre;HA-LepRb flox mouse. Left (a): Cross sections of the dendritic shaft and the spine-like structure are outlined by stippled lines and by yellow transparent color. Right (b): Shown is a cross section of same dendritic shaft, but at a different level. Two mitochondria are labeled (M). B: Left: 3D-reconstruction of the dendritic segment. The location of the two EM microphotographs from A are marked (a and b). Shown is a partially transparent representation with intracellular mitochondria in green and the dendritic surface in gray. Middle: Non-transparent surface representation. Bottom: Shown is a partially transparent representation with shaft synapses depicted in red (N=20). Total length is ~12.5 µm (250 sections – each ~50 nm thick).