Analysis of Transcription Factor mRNAs in Identified Oxytocin and Vasopressin Magnocellular Neurons Isolated by Laser Capture Microdissection

Abstract

The oxytocin (Oxt) and vasopressin (Avp) magnocellular neurons (MCNs) in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON), and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM), and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV) vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun) are differentially expressed in the Oxt and Avp MCNs.

A RNA-Seq Analysis of the Rat Supraoptic Nucleus Transcriptome: Effects of Salt Loading on Gene Expression

“…The brain block containing the hypothalamus was then rapidly frozen on dry ice and stored at −80°C until sectioning was done. Details about this procedure were previously published [ 28 , 29 ]. Briefly, the frozen brain was mounted onto Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA) on a chuck with the caudal portion of the brain placed down on the chuck, and the brain on the chuck was placed in the cryostat (Reichert-Jung 2800, Frigocut, Heidelberg, Germany) for twenty minutes to equilibrate the brain with a chamber temperature (CT) of −20°C and an object temperature (OT) of −16°C.…”

“…# LCM0211). The PEN membrane frame slide was loaded on the instrument with the tissue side down and a clean glass slide underneath it acting as support [ 28 ]. The rat SON is completely visualized in the 20X objective view of the Arcturus XT LCM microscope which allowed for the microdissections of multiple SONs from many serial sections from the same brain specimen onto the same cap.…”

The −216‐ to −100‐bp Sequence in the 5′‐Flanking Region of the Oxytocin Gene Contains a Cell‐Type Specific Regulatory Element for its Selective Expression in Oxytocin Magnocellular Neurones

“…For example, Chu and Zingg showed that a mouse OXT gene promoter transfected into Neuro 2A was activated by ROR α, a member of the ROR/RZR orphan receptor subfamily, produced a six‐fold increase in transcription at two sites (−160 bp and −180 bp) in the CHRE region. In a recent study from our laboratory that investigated whether ROR α was present in the SON, we found that ROR α was indeed significantly present in MCNs but at higher levels in AVP‐MCNs than OXT‐MCNs . Another NHR present in the SON is oestrogen receptor β and, interestingly, its expression is also normally greater in AVP‐MCNs than in OXT‐MCNs and is inversely correlated with the level of expression of the neuropeptide genes .…”

Parvalbumin-Neurons of the Ventrolateral Hypothalamic Parvafox Nucleus Receive a Glycinergic Input: A Gene-Microarray Study

“…Laser-capture microdissection has been used in a variety of publications for the identification of marker genes that may help in the genetic dissection of functionally well characterized regions of the medial hypothalamus (Segal et al, 2005; Paulsen et al, 2009; Landmann et al, 2012; Lee et al, 2012; Humerick et al, 2013). …”