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Comparative Study
. 2010 Sep 2:1350:139-50.
doi: 10.1016/j.brainres.2010.03.082. Epub 2010 Apr 7.

Differential gene expression between neuropeptide Y expressing neurons of the dorsomedial nucleus of the hypothalamus and the arcuate nucleus: microarray analysis study

Shin Draper  1 , Melissa Kirigiti, Maria Glavas, Bernadette Grayson, C N Angie Chong, Betty Jiang, M Susan Smith, Lori M Zeltser, Kevin L Grove
Affiliations
Comparative Study

Differential gene expression between neuropeptide Y expressing neurons of the dorsomedial nucleus of the hypothalamus and the arcuate nucleus: microarray analysis study

Shin Draper et al. Brain Res. .

Abstract

The Dorsomedial Nucleus of the Hypothalamus (DMH) is known to play important roles in ingestive behavior and body weight homeostasis. The DMH contains neurons expressing Neuropeptide Y (NPY) during specific physiological conditions of hyperphagia and obesity, however, the role of DMH-NPY neurons has yet to be characterized. In contrast to the DMH-NPY neurons, NPY expressing neurons have been best characterized in the Arcuate Nucleus of the Hypothalamus (ARH). The purpose of this study is to characterize the chemical phenotype of DMH-NPY neurons by comparing the gene expression profiles of NPY neurons in the DMH and ARH isolated from postnatal NPY-hrGFP mice by microarray analysis. Twenty genes were differentially expressed in the DMH-NPY neurons compared to the ARH. Among them, there were several transcriptional factors that play important roles in the regulation of energy balance. DMH-NPY neurons expressed Glutamic Acid Decarboxylase (GAD) 65 and 67, suggesting that they may be GABAergic, similar to ARH-NPY neurons. While ARH-NPY neurons expressed leptin receptor (ObRb) and displayed the activation of STAT3 in response to leptin administration, DMH-NPY neurons showed neither. These findings strongly suggest that DMH-NPY neurons could play a distinct role in the control of energy homeostasis and are differentially regulated from ARH-NPY neurons through afferent inputs and transcriptional regulators.

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Figures

Figure 1
Figure 1. Microdissection of the DMH and ARH from P16-17 NPY-hrGFP mice
A. Nissl staining of a coronal section. The inserted box shows the location of the DMH and ARH. The right panel illustrates the microdissection technique to obtain isolated DMH and ARH sections from P16-17 NPY-hrGFP mice. B. RT-PCR for Npy, AgRP and Pomc transcript in DMH and ARH-NPY neurons isolated from P16-17 NPY-hrGFP mice. Cyclophilin B was used as an internal control. C. Scatter plot showing DMH-NPY enriched genes (red) and ARH-NPY enriched genes (green) by high stringent analysis. The scale is arbitrary and indicates the level of gene expression in the ARH vs. DMH. The grey dots represent transcripts that are not significantly different between ARH and DMH samples.
Figure 2
Figure 2. Verification of microarray analysis
A. RT-PCR for Foxa1 and Pgc-1α mRNA expression in the DMH and ARH-NPY neurons isolated from P16-17 NPY-hrGFP mice. Cyclophilin B was used an internal control. B. Dark field photomicrograph showing Pgc-1α mRNA localization in the DMH from P16 NPY-hrGFP mouse. C. Fluorescent in situ hybridization for Foxa1 (red) in the DMH (upper) and ARH (lower) of P16 NPY-hrGFP mouse (green). DMHnc: DMH non-compact zone, DMHc: DMH compact zone.
Figure 3
Figure 3. Immunohistochemistry for PGC-1α in P16 NPY-hrGFP mouse
A. 4× objective images of PGC-1α staining in the DMH and ARH. B. Upper panels show 20× confocal images of PGC-1α (red) and overlay image with NPY-GFP (green) in the DMH. Lower panels show 20× confocal images of PGC-1α (red) and overlay image with NPY-GFP (green) in the ARH. The arrows indicate co-localization. C. The statistical analyses showed that 65% of DMH-NPY neurons were co-localized with PGC-1α while only 35% of ARH-NPY neurons were co-localized. (n=3 for each group, P< 0.05 by t-test) DMHnc: DMH non-compact zone, DMHc: DMH compact zone, ME: median eminence.
Figure 4
Figure 4. Immunohistochemistry for FOXA 1 in P16 NPY-hrGFP mouse
A. 4× objective images of FOXA1 staining in the DMH and ARH. B. Upper panels show 20× confocal images of FOXA1 (red) and overlay image with NPY-GFP (green) in the DMH. Lower panels show 20× confocal images of FOXA1 (red) and overlay with NPY-GFP (green) in the ARH. The arrows indicate co-localization. DMHnc: DMH non-compact zone, DMHc: DMH compact zone, ME: median eminence.
Figure 5
Figure 5. Leptin signaling in DMH and ARH-NPY neurons
A. p-STAT3 activation in the DMH (upper panels) and ARH (lower panels) 45min after i.p. 3mg/kg leptin administration in P16 NPY-hrGFP mice. NPY neurons are shown as green and p-STAT3-IR are black nuclear staining. The arrows indicate NPY neurons containing p-STAT3 activation. B. The bar graph shows that only 3-4% NPY positive neurons in the DMH contained p-Stat activation compared to near 70% ARH-NPY neurons (n=3 for each group, p=0.0007 by unpaired t-test). C. RT-PCR for ObRb mRNA expression in the DMH and ARH-NPY neurons isolated from P16-17 NPY-hrGFP mice.
Figure 6
Figure 6. Gad 65 and 67 mRNA expressions in the DMH and ARH-NPY neurons
DMH and ARH-NPY samples were isolated from P16-17 NPY-hrGFP mice.
See this image and copyright information in PMC

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