Gonadotropin-releasing hormone (GnRH) plays a critical role in the central regulation of reproduction. In the absence of GnRH, reproduction is simply not possible in any known mammal. Because GnRH neurons are few in number (about 1000-1200 per brain) and scattered throughout the basal forebrain and hypothalamus, they are very difficult to study at the electrophysiological and molecular levels. To address these limitations, we will produce a transgenic rat in which the enhanced Green Fluorescent Protein (eGFP) is expressed under the control of the rat GnRH promoter, a promoter previously shown to correctly target transgene expression to GnRH neurons. The development of this transgenic rat will allow one to easily identify GnRH neurons by the fluorescent properties of GFP without major tissue treatments and importantly, such identifications are feasible in living cells (e.g. tissue slice preparations). The rat is ideal for such studies due to the vast behavioral, neuroendocrinological and neuroanatomical literature in this species. Further, the rat is an ideal animal model for which a series of surgical, pharmacological and additional treatment procedures relevant to the study of reproduction are well established. We present preliminary data on the design, in vitro analysis of our transgene and document the existence of 13 transgenic founder rats (i.e. contain our transgene in their genome). In this application, we plan to verify that the expression of eGFP is limited to GnRH neurons in this transgenic rat using combined immunocytochemistry and eGFP fluorescence. Given that three separate research groups have used the GnRH promoter to successfully target eGFP, DGal or luciferase transgenes to GnRH neurons in mice, we expect our strategy in rats to be equally effective. Once GFP expression is documented and shown to be GnRH specific, we plan to perform two types of molecular studies. Both of these studies will focus on the evaluation of gene expression in single GnRH neurons using GFP to guide cell identification. These strategies will involve (1) laser capture microscopy and (2) single cell isolation using electrophysiological recording electrodes. We have developed both methods for this application as each has particular strengths and weaknesses. These gene expression studies will initially focus on the identification of receptors (GPCRs) and ion channel (subunit) RNAs known to be expressed within GnRH neurons (as well as several novel RNAs) that will confirm and expand our knowledge of the types of signals capable of modifying GnRH secretion. Taken together these initial characterization studies will be key to validating our transgenic animal prior to embarking on broad evaluations of gene expression patterns within GnRH neurons and electrophysiological studies.
| Thompson, Robert C; Deo, Monika; Turner, David L (2007) Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes. Methods 43:153-61 |
