Visualization of Activated Neurons Involved in Endocrine and Dietary Pathways Using GFP-Expressing Mice
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Neuropeptide Y (NPY) and growth hormone releasing hormone (GHRH) neurons in the hypothalamic arcuate nucleus play an important role in the neuroendocrine control of energy homeostasis and growth hormone secretion. These neuropeptides are synthesized in such small quantities that the common practice was to use colchicine, a neuronal transport inhibitor, to enhance the levels of neuropeptides in the cell body in order to visualize them by immunochemistry. However, colchicine also induces a marked increase in c-Fos levels, suggesting that it affects other mechanisms than solely axonal transport. The development of transgenic mice expressing fluorescent proteins under the control of specific neuropeptide promoters allows direct visualization of specific cell population within the central nervous system. The use of these genetic tools alleviates the need for colchicine pretreatment and can thus be combined with techniques aimed at detecting activated neurons, using the protein c-Fos, as a marker.
In the present article we discuss different variants of green fluorescent protein (GFP), which vary in brightness, cellular localization and can be differentially expressed through the use of different promoters, to visualize GFP-positive neurons. We also address methods to ensure that the GFP fluorescent signal is optimized during additional immunohistochemical procedures. We compared different GFP constructs in which the fluorescence exhibits variable levels of intensity or cellular localization. Recent developments with new GFP variants expressed under the control of strong promoters may provide enhanced and increased stability of fluorescence. Two examples are mice expressing an enhanced GFP protein in GHRH neurons and the NPY-Renilla-GFP mouse, in which a long bacterial artificial chromosome sequence as a promoter and a free cytoplasmic GFP renders the signal more stable and brighter than other variants of GFPs. In these examples GFP expression is localized mostly in cell bodies, and these two models are advantageous for studies investigating the phenotypical identity of neurons activated by physiological stimuli, like ghrelin. Differential subcellular expression of GFP is highlighted by the NPY-Tau-Sapphire-GFP mice in which the blue-shifted GFP variant (Sapphire-GFP) is fused to the tau sequence, allowing its cellular targeting to axon terminals thereby making this model suitable for studies investigating neuronal terminals.
