Use of Laser-Capture Microdissection for the Identification of Marker Genes for the Ventromedial Hypothalamic Nucleus

Abstract

The ventromedial hypothalamic nucleus (VMH) plays an important role in the control of feeding and energy homeostasis. In contrast to other hypothalamic nuclei that are also known to regulate energy balance, there is a paucity of nucleus-specific marker genes for the VMH, limiting the application of molecular approaches for analyzing VMH information processing, function, and circuitry. Here, we report the use of laser-capture microdissection to isolate a set of cDNAs that are enriched in the VMH relative to two adjacent hypothalamic nuclei, the arcuate and dorsomedial hypothalamus. The relative expression levels of nine of the 12 most robustly expressed VMH-enriched genes were confirmed by real-time PCR analysis using separate RNAs from these three nuclei. Three of these VMH-enriched genes were further characterized by in situ hybridization histochemistry, including pituitary adenylate cyclase activating polypeptide, cerebellin 1, and an expressed sequence tag named LBH2. Finally, to test whether some of these genes were coordinately regulated, we monitored their expression in steroidogenic factor 1 (SF-1) knock-out mice. SF-1 is a transcription factor that controls the development of the VMH. The RNA levels for four of these genes were reduced in these knock-out animals, further suggesting that they are direct or indirect targets of this orphan nuclear receptor. The VMH-enriched genes identified here provide a basis for a functional analysis of VMH neuronal subpopulations via the use of bacterial artificial chromosome transgenics and related technologies. These results also demonstrate the utility of laser-capture microdissection coupled with microarray technology to identify nucleus-specific transcriptional networks.

Delayed Satiety-Like Actions and Altered Feeding Microstructure by a Selective Type 2 Corticotropin-Releasing Factor Agonist in Rats: Intra-Hypothalamic Urocortin 3 Administration Reduces Food Intake by Prolonging the Post-Meal Interval

“…Although this simplistic view of the VMN fell into disfavor (Teitelbaum, 1957;Peters et al, 1978), the VMN has been rediscovered as a receptive site for short-and long-term energy feedback signals and forms an integrated component of energy balance neurocircuitry (Zaborszky and Makara, 1979;Luiten and Room, 1980;Ter Horst and Luiten, 1987). Rats with impaired VMN development secondary to deficiency of steroidogenic factor-1, a specific marker of VMN neurons (Segal et al, 2005), are hyperphagic, less active and develop adult-onset obesity (Majdic et al, 2002). The dorsomedial VMN, where injections in the present study were targeted, is Figure 7 Illustration of the serial regularity within feeding bouts after bilateral injection of murine Ucn 3 into the paraventricular nucleus of the hypothalamus.…”

Leptin responsiveness restored by amylin agonism in diet-induced obesity: Evidence from nonclinical and clinical studies

“…4 A and C-F). To clarify the phenotype of leptin-responsive VMH neurons in amylin-pretreated rats, we assessed whether pSTAT3 was colocalized with steroidogenic factor-1 (SF-1), a highly enriched protein in the VMH implicated in weight regulation (21,22). A moderate population of VMH cells exhibited both pSTAT3 and SF-1 labeling after leptin administration (Fig.…”

PACAP Neurons in the Hypothalamic Ventromedial Nucleus Are Targets of Central Leptin Signaling

“…We investigated the metabolic role of PACAP, a pleiotropic neuropeptide, which was recently identified by microarray as a "VMN-enriched" gene (Segal et al, 2005;Kurrasch et al, 2007).…”

“…Like many hypothalamic neurons, at least some of the effects of SF-1 cells in the VMN are mediated by glutamate (Tong et al, 2007), although neuropeptidergic classifications remain elusive. Microarray studies have identified a number of VMN-enriched genes, including the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) (Segal et al, 2005;Kurrasch et al, 2007), which has been implicated in many central functions (for review, see Vaudry et al, 2000). The pleiotropic nature of this peptide has made it difficult to study in terms of energy balance; however, in this study, we have shown by in vivo pharmacological and histological methods that the VMN PACAP population partially mediates responses to leptin, and that PACAP exerts robust effects on both appetite and energy expenditure in mice.…”