RNAi ? a new targeted silencer?

Gene silencing causes marked behavior changes, may help map brain circuitry

By | June 27, 2006

For the first time, researchers have used RNA interference (RNAi) to genetically silence estrogen's effects in a specific part of the mouse brain, rather than throughout the entire body. The technique, reported online June 26 in the Proceedings of the National Academy of Sciences, led to striking changes in female sexual behavior, and could help scientists map brain circuitry, according to the authors. The results show that RNAi "can really help map out the genes and neural networks behind complex behaviors," co-author Sonoko Ogawa at Rockefeller University in New York told The Scientist. In the past, scientists have generated female mice that completely lack estrogen receptors alpha or beta. Estrogen receptor alpha knockout mice aggressively reject male advances; beta knockout mice, in contrast, exhibit normal sexual behavior. Experiments using global knockout mice can't distinguish between the effects of estrogen in adulthood and earlier in development, according to study co-author Donald Pfaff at Rockefeller University. Specifically, it's unclear whether estrogen receptor alpha knockout mice exhibit strange behavior because they need the hormone's effects for mating, or because the receptor was absent during brain development, when estrogen plays a key role, Pfaff told The Scientist. To pinpoint estrogen receptor alpha's role, Pfaff, Ogawa, and colleagues used RNAi to knock out the receptor's expression in female adult mice in the ventromedial nucleus of the hypothalamus, a brain region involved in female reproductive behavior. The researchers injected an adeno-associated viral vector carrying a small hairpin targeting estrogen receptor alpha into the ventromedial nucleus of female mice. Immunohistochemistry analysis confirmed targeted cells no longer expressed the receptor but did not die, and estrogen receptor alpha expression remained present elsewhere in the brain and body. The mice that received the RNAi treatment were not receptive to males. Instead, they aggressively rejected male approaches or attempts to mount them with kicks, acting similarly to estrogen receptor alpha knockout mice -- suggesting that estrogen has its effect on reproductive behavior during adulthood, not development. Previous studies have tried to use antisense techniques to suppress estrogen receptor alpha in specific parts of the adult mouse brain, but these did not completely knock out the receptor or proved toxic, Ogawa explained. "Using RNAi, we can completely knock out the receptor in a specific part of the brain," she noted. Ogawa and her colleagues are currently investigating other behavioral effects of knocking out estrogen receptor alpha in the ventromedial nucleus, which also plays a role in food intake and body weight. A future experiment could test the behavioral effects of RNAi against estrogen receptor beta, Pfaff added. Based on where estrogen receptor beta is expressed in the brain, such as the amygdala and the cerebral cortex, "we expect to have effects on fear conditioning and in cognitive activities," respectively, he said. Emilie Rissman at the University of Virginia in Charlottesville, who did not participate in this study, called the findings "a good technical advance." It makes sense to move on to areas of the brain where the functions of receptors are less clear, she added. "There are regions of the brain such as the bed nucleus of the stria terminalis, which is important for investigatory behavior and male copulatory behavior, where estrogen receptor alpha and estrogen receptor beta are both present. Using RNAi could help unmuddy our interpretation of which receptor is responsible for which of these behaviors," she told The Scientist. Estrogen receptors are involved in the sexual differentiation of the brain, "and it would be neat now to do developmental studies that knock down the receptors early in life in specific brain areas to see which are important for sexual differentiation," Larry Young at Emory University in Atlanta, also not a co-author, told The Scientist. Charles Q. Choi cchoi@the-scientist.com Links within this article G. Dutton and J.M. Perkel. "Shhh: Silencing genes with RNA interference." The Scientist, April 7, 2003. www.the-scientist.com/article/display/13678/ S. Musatov et al. "RNAi-mediated silencing of estrogen receptor in the ventromedial nucleus of hypothalamus abolishes female sexual behaviors." PNAS, published online ahead of print June 26, 2006. www.pnas.org J.U. Adams. "Scratching the surface for estrogen's effects." The Scientist, July 5, 2004. www.the-scientist.com/article/display/14801/ Donald Pfaff www.rockefeller.edu/labheads/pfaff/pfaff-lab.php A. Nicot, D.W. Pfaff. "Antisense oligodeoxynucleotides as specific tools for studying neuroendocrine and behavioral functions: Some prospects and problems." Journal of Neuroscience Methods, January 1997. PM_ID: 9125374 Emilie Rissman www.virginia.edu/biology/develbio/faculty/rissman.htm Larry Young www.yerkes.emory.edu/YOUNG/

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