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Science 334 (6059): 1133-1137

Copyright © 2011 by the American Association for the Advancement of Science

Transplanted Hypothalamic Neurons Restore Leptin Signaling and Ameliorate Obesity in db/db Mice

Artur Czupryn1,2,3,*, Yu-Dong Zhou4,*,{dagger}, Xi Chen5,*, David McNay5,{ddagger}, Matthew P. Anderson4,||,, Jeffrey S. Flier5,6,§,||, and Jeffrey D. Macklis1,2,6,§,||

1 Department of Stem Cell and Regenerative Biology, and Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.
2 MGH-HMS Center for Nervous System Repair, Departments of Neurology and Neurosurgery, and Program in Neuroscience, Harvard Medical School; Nayef Al-Rodhan Laboratories, Massachusetts General Hospital, Boston, MA 02114, USA.
3 Nencki Institute of Experimental Biology, Department of Molecular and Cellular Neurobiology, Warsaw, 02-093, Poland.
4 Departments of Neurology and Pathology, and Program in Neuroscience, Harvard Medical School; Center for Life Sciences, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
5 Beth Israel Deaconess Medical Center, Division of Endocrinology, Diabetes, and Metabolism, and Harvard Medical School, Boston, MA 02215, USA.
6 Harvard Medical School, Boston, MA 02215, USA.


Figure 1
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Fig. 1. Transplanted E13.5 eGFP+ hypothalamic cells mature into appropriate hypothalamic neuron subtypes. (A) Ultrasound-guided microtransplantation of dissociated E13.5 hypothalamic cells into early postnatal db/db mouse hypothalamus in sagittal view. The ~100-μm-diameter pulled glass micropipette (MP) appears on the ultrasonogram much larger than its real size due to ultrasound shadow. (B) Schematic of a coronal brain section magnified in C. (C) Location and survival of transplanted eGFP+ cells in the representative recipient hypothalamus 20 weeks after microtransplantation. (D and E) Representative confocal three-dimensional (3D) reconstructions of donor-derived eGFP+ neurons in the VMH of db/db mice 20 weeks after microtransplantation, showing (D) a POMC neuron identified by the cleavage product β-endorphin and (E) a NPY neuron. For each confocal reconstruction, the x-z plane is shown at the bottom and the y-z plane is shown at the right. (F to I) Four distinct electrophysiological donor-derived eGFP+ neuron subtypes. (F) The firing behavior of a representative newly integrated eGFP+ donor-derived NRRS neuron. "Type A" and "type C" neurons can be distinguished by the absence or presence, respectively, of delayed firing (bottom traces). Note the delayed firing in "type C" neurons (arrow) when hyperpolarizing current is withdrawn. (G) Representative donor-derived eGFP+ RRS neuron. An apparent voltage sag (dashed line) and a single rebound action potential were typical. (H) Representative donor-derived eGFP+ BS neuron. A low-threshold calcium spike and cluster of rebound action potentials were typical in response to hyperpolarizing current injection. (I) Representative donor-derived eGFP+ FS neuron. A high firing frequency and absent firing adaptation are two typical characteristics of this neuron type. DS, dorsum sellae (bone); ST, sella turcica (bone); AC, anterior commissure; S, skin and fur on the head; 3V, third ventricle. Asterisks demarcate the rostrocaudal extent of the transplantation tracks. Scale bars in (A), 1 mm; in (C), 100 μm; in (D) to (I), 10 μm.

 

Figure 2
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Fig. 2. Transplanted eGFP+ hypothalamic neurons form excitatory and inhibitory synaptic connections and response to the energy state signals leptin, glucose, and insulin. (A and B) Donor-derived eGFP+ neurons in db/db recipients bear extensive appositions with synaptophysin-positive puncta (red). (B) Higher-magnification confocal image of the boxed region of a dendrite in the merged confocal image in (A), showing synaptophysin-positive puncta indicating synaptic contacts. (C) Spontaneous EPSCs and IPSCs were observed in eGFP+ neurons. (D) Representative dual whole-cell recording showing that stimulation of donor-derived eGFP+ neurons (green traces) elicits EPSPs (upper) or IPSPs (lower) in native neurons (black traces). (E) Representative confocal 3D reconstruction of donor-derived eGFP+ neuron in the VMH of db/db mice 20 weeks after microtransplantation with leptin-induced signaling indicated by phosphorylated STAT3 in response to leptin administration. (Bottom) x-z plane; (right) y-z plane. (F) Representative voltage tracing from a leptin-depolarized eGFP+ neuron. Leptin depolarized the membrane potential and substantially increased the firing rate; insulin had the opposite effect. (G) Representative voltage tracing from a leptin-hyperpolarized eGFP+ neuron. Both leptin and insulin hyperpolarized the membrane potential and substantially decreased the firing rate; low glucose had the opposite effect. Scale bars in (A), 8 μm; in (B), 2 μm; in (E), 10 μm.

 

Figure 3
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Fig. 3. Transplanted E13.5 wild-type immature hypothalamic neurons reduce body weight, fat mass, serum leptin, and glucose levels in leptin receptor–deficient db/db mice. (A) Hypothalamically chimeric db/db mice (db/db mice microtransplanted with E13.5 hypothalamic cells into the medial hypothalamus) have significantly lower body-weight gain than nonoperated db/db mice or sham-operated db/db mice (F2,361 = 120.75; chimeric db/db versus db/db nonoperated mice, P < 0.001; chimeric db/db versus db/db sham-operated mice, P < 0.001). Microtransplantation of E13.5 hypothalamic cells into nonobese control mice did not change body weight compared with nonobese, nonoperated littermates. (B) Analysis of body composition using DEXA demonstrates significant fat mass reduction in transplanted compared with nontransplanted obese (db/db) mice. (C and D) Analysis of blood parameters demonstrates statistically significant reductions in (C) serum leptin levels at 13 weeks of age and (D) blood glucose at 9 and 13 weeks of age. *P < 0.05; **P < 0.01; ***P < 0.001. Data reported as mean ± SEM.

 


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