Identification of Kappa-Opioid Receptor-Expressing Cells in the Hypothalamus with Transgenic Rats

Presentation Number: SUN 471
Date of Presentation: April 2nd, 2017

Chudai Takahashi*1, Teppei Goto2, Masumi Hirabayashi3, Sho Nakamura4, Mingdao Dai1, Kana Ikegami5, Fuko Matsuda1, Yoshihisa Uenoyama5, Hiroko Tsukamura5 and Kei-ichiro Maeda4
1Department of Veterinary Medical Sciences, The University of Tokyo, Japan, 2National Institute for Physiological Sciences, 3National Institute for Physiological Sciences, Japan, 4Department of Veterinary Medical Sciences, The University of Tokyo, Tokyo, Japan, 5Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

Abstract

Kisspeptin neuronal populations are localized in the arcuate nucleus and anteroventral periventricular nucleus to regulate gonadotropin-releasing hormone (GnRH) secretion in rats. The arcuate kisspeptin neurons coexpress neurokinin B (NKB) and dynorphin (Dyn) and, therefore, are referred as KNDy neurons. Since NKB stimulates and Dyn suppresses GnRH release, the interaction of these neuropeptides are involved in generating GnRH pulses. The NK3R, a NKB receptor, is localized in the KNDy neurons, but the kappa-opioid receptor (KOR), a Dyn receptor-expressing cells are yet unidentified.

 The present study generated a transgenic rat, in which KOR-expressing cells are visualized by Venus, a fluorescent protein to identify KOR-expressing cells in the brain. The transgene consisting of KOR promoter gene fused with Venus gene at the 3’ end was introduced to rat fertilized eggs. Venus expression in the transgenic rats were analyzed using ovariectomized and estradiol-treated mature females. The expression of Oprk1 mRNA was detected by in situ hybridization. The protein expressions of Venus and other cell markers were detected with immunohistochemistry. Most Oprk1 mRNA-expressing cells showed Venus-immunoreactivities, suggesting that KOR-positive cells were successfully visualized with Venus in the transgenic rats. Venus-immunopositive cells were found in the cerebral cortex and paraventricular, dorsomedial and arcuate nuclei of the hypothalamus. The distribution of Venus protein is consistent with that in the previous studies showing KOR mRNA expressions. Cell types of Venus-expressing cells were identified with dual immunohistochemistry of Venus and either NeuN, a neuronal marker, kisspeptin or ERα. The dual immunohistochemistry showed that most of the Venus-positive cells expressed NeuN in every nuclei examined. On the other hand, most of the Venus-positive cells did not express kisspeptin. Some Venus-positive cells expressed ERα in the arcuate nucleus. The present result suggests that most of KOR-expressing cells are neurons but not KNDy neurons. Since Dyn-KOR signaling is involved in GnRH pulse generation, the arcuate non-kisspeptidergic KOR neurons would be a part of the GnRH pulse-generating system. The transgenic rat, therefore, would be useful to identify the gene cluster expressed in KOR-expressing cells.

 

Nothing to Disclose: CT, TG, MH, SN, MD, KI, FM, YU, HT, KIM