Research Article: New Research | Integrative Systems Arcuate and Preoptic Kisspeptin neurons exhibit differential projections to hypothalamic nuclei and exert opposite postsynaptic effects on hypothalamic paraventricular and dorsomedial nuclei in the female mouse https://doi.org/10.1523/ENEURO.0093-21.2021 Cite as: eNeuro 2021; 10.1523/ENEURO.0093-21.2021 Received: 10 March 2021 Revised: 21 June 2021 Accepted: 11 July 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 Stincic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Title page 2 Manuscript Title: Arcuate and Preoptic Kisspeptin neurons exhibit differential projections to hypothalamic nuclei 3 and exert opposite postsynaptic effects on hypothalamic paraventricular and dorsomedial nuclei in the female 4 mouse 5 Abbreviated title: Projections and functions of kisspeptin neurons 6 Authors: Todd L. Stincic1, Jian Qiu1, Ashley M. Connors1a, Martin J. Kelly1,2, Oline K. Rønnekleiv1,2 7 1Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, 8 Oregon 97239; 2Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and 9 Science University, Beaverton, Oregon 97006. aCurrent address: Department of Biological Sciences, North 10 Carolina State University, Raleigh, NC 27695. 11 Author contribution: O.K.R. and M.J.K. designed the research; T.L.S, J.Q, A.C, and O.K.R. performed research 12 and analyzed data. O.K.R., T.L.S. and M.J.K., wrote the paper with input from the other authors. 13 *Corresponding Author: Oline K. Rønnekleiv, Department of Chemical Physiology and Biochemistry, Oregon 14 Health and Science University, Portland, Oregon 97239; Email: [email protected] 15 Number of figures: 18 16 Number of Tables: 3 17 Number of words in Abstract: 250 18 Number of words in Significance: 120 19 Number of words in Introduction: 689 20 Number of words in Discussion: 2431 21 Acknowledgements: We thank Ms. Martha A. Bosch for excellent technical support and assistance with 22 creating the figures. We also thank Mr. Dan Johnson for excellent help with maintaining the mice breeding 23 colony including doing the genotyping of mice. Finally, we would like to thank Dr. Stefanie Petrie Kaech and 24 Mr. Brian Jenkins for assistance with confocal imaging. 25 Conflict of Interest: The authors declare no competing financial interests. 26 Funding sources: This work was supported by National Institutes of Health Grants R01-NS043330 (OKR), 27 R01-DK068098 (OKR & MJK) and a grant from OHSU Center for Women’s Health Circle of Giving (OKR & 28 MJK). Confocal microscopy was supported by P30 NS061800 grant (PI, S. Aicher) 29 1 30 Abstract 31 Kisspeptin (Kiss1) neurons provide indispensable excitatory input to GnRH neurons, which is important for the 32 coordinated release of gonadotropins, estrous cyclicity and ovulation. However, Kiss1 neurons also send 33 projections to many other brain regions within and outside the hypothalamus. Two different populations of 34 Kiss1 neurons, one in the arcuate nucleus (Kiss1ARH) and another in the anteroventral periventricular and 35 periventricular nucleus (Kiss1AVPV/PeN) of the hypothalamus are differentially regulated by ovarian steroids, and 36 are believed to form direct contacts with GnRH neurons as well as other neurons. To investigate the projection 37 fields from Kiss1AVPV/PeN and Kiss1ARH neurons in female mice, we used anterograde projection analysis, and 38 channelrhodopsin-assisted circuit mapping (CRACM) to explore their functional input to select target neurons 39 within the paraventricular (PVH) and dorsomedial (DMH) hypothalamus, key pre-autonomic nuclei. Cre- 40 dependent viral (AAV1-DIO-ChR2 mCherry) vectors were injected into the brain to label the two Kiss1 neuronal 41 populations. Immunocytochemistry for mCherry and neuropeptides combined with confocal microscopy was 42 used to determine the projection-fields of both Kiss1 neuronal groups. Whole-cell electrophysiology and 43 optogenetics was used to elucidate the functional input to the PVH and DMH. Our analysis revealed many 44 common, but also several clearly separate projection fields between the two different populations of Kiss1 45 neurons. In addition, optogenetic stimulation of Kiss1 projections to PVH prodynorphin, Vglut2 and DMH 46 CART-expressing neurons, revealed excitatory glutamatergic input from Kiss1ARH neurons and inhibitory 47 GABAergic input from Kiss1AVPV/PeN neurons. Therefore, these steroid-sensitive Kiss1 neuronal groups can 48 differentially control the excitability of target neurons to coordinate autonomic functions with reproduction. 49 Words: 250 50 Significance Statement 51 Hypothalamic kisspeptin (Kiss1) neurons are the most gonadal steroid-sensitive neurons in the brain, and its 52 principle neurotransmitter kisspeptin is essential for sexual development and reproduction through direct 53 excitation of GnRH neurons. Kiss1 neurons also co-express either the classical neurotransmitters GABA, 54 which we document is released only by Kiss1AVPV/PeN neurons or glutamate, which is released by Kiss1ARH 55 neurons. Consequently, Kiss1AVPV/PeN neurons have direct inhibitory and Kiss1ARH neurons direct excitatory 56 actions onto PVH and DMH neurons known to controlling food intake and energy expenditure, respectively. 57 Therefore, we have found that Kiss1 neurons have a significant input to “pre-autonomic” neurons known to 58 regulate multiple homeostatic functions, which would help coordinate reproduction with these other functions 59 that are vital for survival of the species. 60 Words: 120 61 62 2 63 Introduction 64 Early in this century it was discovered that that mutations in an orphan receptor GPR54 caused hypothalamic 65 hypogonadism in humans, and deletion of GPR54 in mice resulted in defective sexual development and 66 reproductive failure (De Roux et al., 2003; Seminara et al., 2003; d'Anglemont de Tassigny et al., 2007). It was 67 subsequently discovered that a fragment of the peptide metastin, kisspeptin (Kiss1), was expressed in the 68 hypothalamus and deletion of Kiss1 also caused hypothalamic hypogonadism (d'Anglemont de Tassigny et al., 69 2007). Deletion of GPR54 also results in obesity in female mice (Tolson et al., 2014). The distribution and 70 gonadal steroid regulation of hypothalamic Kiss1 neurons was quickly determined in numerous species 71 including human for review see (Smith, 2008; Oakley et al., 2009; Lehman et al., 2013; Hrabovszky, 2014). 72 Neurons expressing Kiss1 are located primarily in two distinct areas of the forebrain: The preoptic area (POA) 73 and the basal hypothalamus. The preoptic Kiss1 populations in rodents are located in the anteroventral 74 periventricular (AVPV; Kiss1AVPV) and adjacent periventricular nuclei (PeN; Kiss1PeN) and these neurons co- 75 express tyrosine hydroxylase (TH), vesicular GABA transporter (vGat) and in some animal models also 76 vesicular glutamate transporter-2 (vGlut2) (Cravo et al., 2011; Zhang et al., 2013). Importantly, these rostral 77 Kiss1AVPV/PeN neurons are positively regulated by 17β-estradiol (E2) (Smith et al., 2005; Zhang et al., 2013). 78 The basal hypothalamic Kiss1 population is located in the arcuate nucleus of the hypothalamus (ARH; 79 Kiss1ARH) with scattered neurons also in the hypothalamic dorsomedial nucleus (DMH) (Clarkson et al., 2009; 80 Bosch et al., 2012). The mRNA expression of the neuropeptides, including Kiss1, NKB (tac2) and prodynorphin 81 (pDyn) within the Kiss1ARH neurons are all inhibited by E2, whereas vGlut2 mRNA, also expressed in Kiss1ARH 82 neurons, and glutamate release are increased by E2 in females (Navarro et al., 2009; Qiu et al., 2018). The 83 Kiss1AVPV/PeN neurons send direct projections onto gonadotropin-releasing hormone (GnRH) neurons and are 84 essential for positive feedback regulation of GnRH and LH secretion (Clarkson et al., 2008; Yip et al., 2015; 85 Qiu et al., 2016; Piet et al., 2018). Kiss1ARH neurons in mice do not appear to contact GnRH cell bodies, but 86 form close anatomical contacts with distal GnRH nerve processes and also exhibit neurophysiological 87 (functional) interaction with Kiss1AVPV/PeN neurons (Yip et al., 2015; Qiu et al., 2016). Later it was discovered 88 that Kiss1ARH neurons project to Proopiomelanocortin and Neuropeptide Y/Agouti related peptide (NPY/AgRP) 89 neurons, which suggested that Kiss1 neurons may also be involved in regulating feeding behaviors (Qiu et al., 90 2018; Padilla et al., 2019). Kisspeptin immunoreactive fibers are located in many different brain regions 91 including median preoptic nucleus (MnPO), ventral lateral septum nucleus (LSV), bed nucleus of the stria 92 terminalis (BST), paraventricular nucleus of the hypothalamus (PVH), supraoptic nucleus (SON), lateral 93 hypothalamus (LH) and dorsomedial hypothalamic (DMH) nucleus, suggesting widespread projections of Kiss1 94 neurons (Clarkson et al., 2009; Marraudino et al., 2017). However, with the exception of Kiss1 input to GnRH 95