Laser-capture microdissection and transcriptional profiling of the dorsomedial nucleus of the hypothalamus

Department of Internal Medicine and Department of Pharmacology, Division of Hypothalamic Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9077.
The Journal of Comparative Neurology (Impact Factor: 3.23). 11/2012; 520(16):3617-32. DOI: 10.1002/cne.23116
Source: PubMed


Identifying neuronal molecular markers with restricted patterns of expression is a crucial step in dissecting the numerous pathways and functions of the brain. While the dorsomedial nucleus of the hypothalamus (DMH) has been implicated in a host of physiological processes, current functional studies have been limited by the lack of molecular markers specific for DMH. Identification of such markers would facilitate the development of mouse models with DMH-specific genetic manipulations. Here we used a combination of laser-capture microdissection (LCM) and gene expression profiling to identify genes that are highly expressed within the DMH relative to adjacent hypothalamic regions. Six of the most highly expressed of these genes, Gpr50, 4930511J11Rik, Pcsk5, Grp, Sulf1, and Rorβ, were further characterized by real-time polymerase chain reaction (PCR) analysis and in situ hybridization histochemistry. The genes identified in this article will provide the basis for future gene-targeted approaches for studying DMH function. J. Comp. Neurol. 520:3617-3632, 2012. © 2012 Wiley Periodicals, Inc.

Download full-text


Available from: Syann Lee, Sep 09, 2014
1 Follower
35 Reads
  • Source
    • "( Figure 6A ) . However , PPARγ expression in the hypothalamus was 140 - fold lower than that in the BAT . To determine which hypothalamic nuclei accounted for the detected expression , we analyzed PPARγ expression in laser - capture microdissected hypothalamic nuclei according to the methods we have previously utilized ( Bookout et al . , 2006b ; Lee et al . , 2012 ) . In agreement with our ISH results , the hypothalamic site that exhibited the highest level of expression was the SCh ( Figure 6B ) . PPARγ was also detectable in the ARC , albeit at a low level ( mean Ct ∼29 . 2 ) . In all other examined hypothalamic nuclei , PPARγ was near the limit of detection ( mean Ct >30 ) ( Figure 6B ) . Beca"
    [Show abstract] [Hide abstract]
    ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor that was originally identified as a regulator of peroxisome proliferation and adipocyte differentiation. Emerging evidence suggests that functional PPARγ signaling also occurs within the hypothalamus. However, the exact distribution and identities of PPARγ-expressing hypothalamic cells remains under debate. The present study systematically mapped PPARγ mRNA expression in the adult mouse brain using in situ hybridization histochemistry. PPARγ mRNA was found to be expressed at high levels outside the hypothalamus including the neocortex, the olfactory bulb, the organ of the vasculosum of the lamina terminalis (VOLT), and the subfornical organ. Within the hypothalamus, PPARγ was present at moderate levels in the suprachiasmatic nucleus (SCh) and the ependymal of the 3rd ventricle. In all examined feeding-related hypothalamic nuclei, PPARγ was expressed at very low levels that were close to the limit of detection. Using qPCR techniques, we demonstrated that PPARγ mRNA expression was upregulated in the SCh in response to fasting. Double in situ hybridization further demonstrated that PPARγ was primarily expressed in neurons rather than glia. Collectively, our observations provide a comprehensive map of PPARγ distribution in the intact adult mouse hypothalamus.
    Frontiers in Neuroanatomy 09/2015; 9:120. DOI:10.3389/fnana.2015.00120 · 3.54 Impact Factor
  • Source
    • "Both VM and DM are heterogeneous entities composed of different cell types whose identity and spatial distribution have not been fully resolved yet. Various chemo-and genoarchitectonic mappings suggest in both cases a fundamental organization into compact core portions and surrounding dispersed shell domains (Milhouse 1973; Chou et al. 2001; Choi et al. 2005; Segal et al. 2005; McClellan et al. 2006; Lee et al. 2012; Puelles et al. 2012). The latter authors noted that the VM and DM core domains are nearly completely excitatory (glutamatergic), whereas their shell regions, VMs, DMs, contain more dispersed inhibitory GABAergic neurons intermixed with glutamatergic ones (Puelles et al. 2012; their Figs. "
    [Show abstract] [Hide abstract]
    ABSTRACT: According to the updated prosomeric model, the hypothalamus is subdivided rostrocaudally into terminal and peduncular parts, and dorsoventrally into alar, basal, and floor longitudinal zones. In this context, we examined the ontogeny of peptidergic cell populations expressing Crh, Trh, and Ghrh mRNAs in the mouse hypothalamus, comparing their distribution relative to the major progenitor domains characterized by molecular markers such as Otp, Sim1, Dlx5, Arx, Gsh1, and Nkx2.1. All three neuronal types originate mainly in the peduncular paraventricular domain and less importantly at the terminal paraventricular domain; both are characteristic alar Otp/ Sim1-positive areas. Trh and Ghrh cells appeared specifically at the ventral subdomain of the cited areas after E10.5. Additional Ghrh cells emerged separately at the tuberal arcuate area, characterized by Nkx2.1 expression. Crh-positive cells emerged instead in the central part of the peduncular paraventricular domain at E13.5 and remained there. In contrast, as development progresses (E13.5– E18.5) many alar Ghrh and Trh cells translocate into the alar subparaventricular area, and often also into underlying basal neighborhoods expressing Nkx2.1 and/or Dlx5, such as the tuberal and retrotuberal areas, becoming partly or totally depleted at the original birth sites. Our data correlate a topologic map of molecularly defined hypothalamic progenitor areas with three types of specific neurons, each with restricted spatial origins and differential migratory behavior during prenatal hypothalamic development. The study may be useful for detailed causal analysis of the respective differential specification mechanisms. The postulated migrations also contribute to our understanding of adult hypothalamic complexity.
    Brain Structure and Function 05/2014; 219(3):1083-1111. DOI:10.1007/s00429-013-0554-2 · 5.62 Impact Factor
  • Source
    • "Endocannabinoids and NO that are co-released from DMH neurons differentially regulate GABAergic inhibitory tone and fasting reinforces NO-mediated enhancement of GABAergic currents [14]. Although a recent study further identifies genes that are highly expressed in the DMH using microarray analysis [15], little information is available about molecular markers specific for the DMH, which would facilitate the development of mouse models with DMH-specific genetic manipulations. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The dorsomedial nucleus of the hypothalamus (DMH) contributes to the regulation of overall energy homeostasis by modulating energy intake as well as energy expenditure. Despite the importance of the DMH in the control of energy balance, DMH-specific genetic markers or neuronal subtypes are poorly defined. Here we demonstrate the presence of cholinergic neurons in the DMH using genetically modified mice that express enhanced green florescent protein (eGFP) selectively in choline acetyltransferase (Chat)-neurons. Overnight food deprivation increases the activity of DMH cholinergic neurons, as shown by induction of fos protein and a significant shift in the baseline resting membrane potential. DMH cholinergic neurons receive both glutamatergic and GABAergic synaptic input, but the activation of these neurons by an overnight fast is due entirely to decreased inhibitory tone. The decreased inhibition is associated with decreased frequency and amplitude of GABAergic synaptic currents in the cholinergic DMH neurons, while glutamatergic synaptic transmission is not altered. As neither the frequency nor amplitude of miniature GABAergic or glutamatergic postsynaptic currents is affected by overnight food deprivation, the fasting-induced decrease in inhibitory tone to cholinergic neurons is dependent on superthreshold activity of GABAergic inputs. This study reveals that cholinergic neurons in the DMH readily sense the availability of nutrients and respond to overnight fasting via decreased GABAergic inhibitory tone. As such, altered synaptic as well as neuronal activity of DMH cholinergic neurons may play a critical role in the regulation of overall energy homeostasis.
    PLoS ONE 04/2013; 8(4):e60828. DOI:10.1371/journal.pone.0060828 · 3.23 Impact Factor
Show more