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.
Highlights
The central nervous system is composed of many cellular phenotypes which play diverse and distinct roles in brain functions [1,2,3]
We describe a strategy for the enrichment of specific neuronal phenotypes from the central nervous system (CNS) for the purpose of elucidating their distinct molecular properties
By transducing these neurons using AAVs containing cell-type specific promoters driving he expression of GFP, there is a selective expression of fluorescence only in the specific neuronal phenotypes
Summary
The central nervous system is composed of many cellular phenotypes which play diverse and distinct roles in brain functions [1,2,3]. In order to understand the mechanisms that lead to the development and maintenance of these specific cellular phenotypes it is first necessary to have methods that allow for the enrichment and purification of specific cell phenotypes from the heterogeneous populations of cell-types that exist in the brain This critical step is a prerequisite for the analysis of the unique molecular properties of specific neurons of interest. The neurohypophysial peptide hormones, Oxt and Avp, are best known for their roles in the periphery to stimulate milk let-down during lactation and induce uterine contractions during parturition, and to regulate systemic salt and water balance, respectively [2,7,8] More recently, these neuropeptides have been shown to have important physiological functions and behavioral effects in the central nervous system [2,9,10,11,12,13]
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