Research Article: New Research | Sensory and Motor Systems Topographically distinct projection patterns of early- and late-generated projection neurons in the mouse olfactory bulb https://doi.org/10.1523/ENEURO.0369-20.2020 Cite as: eNeuro 2020; 10.1523/ENEURO.0369-20.2020 Received: 23 August 2020 Revised: 11 October 2020 Accepted: 16 October 2020 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 © 2020 Chon 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: 2 Topographically distinct projection patterns of early- and late-generated projection 3 neurons in the mouse olfactory bulb 4 5 Abbreviated title: 6 Birthdate-dependent classification of mitral cells 7 8 Authors: 9 Uree Chon1, Brandon J. LaFever2, Uyen Nguyen2, Yongsoo Kim1#, and Fumiaki Imamura2# 10 11 Author affiliations: 12 Departments of 1Neural and Behavioral Sciences and 2Pharmacology, Penn State College of 13 Medicine, Hershey PA, 17033, USA 14 15 Author Contributions: 16 YK and FI Designed Research; UC, UN and FI Performed Research; UC, BJL, YK, and FI 17 Analyzed data; UC, BJL, YK, and FI Wrote the paper. 18 19 20 #Corresponding authors: 21 Fumiaki Imamura, Ph.D. 22 Department of Pharmacology, R130 23 Penn State College of Medicine 24 500 University Dr. 25 Hershey, PA 17033 26 Telephone: 717.531.5734 27 FAX: 717.531.5013 28 Email: [email protected] 29 30 Yongsoo Kim, Ph.D. 31 Department of Neural and Behavioral Sciences 32 Penn State College of Medicine 33 500 University Dr. 34 Hershey, PA 17033 35 Email: [email protected] 36 37 38 Number of Figures: 4; Tables: 0; Multimedia: 0 39 40 Number of words for Abstract: 250; Significance statement:118: Introduction: 749; 41 Discussion: 1638. 42 43 44 Conflicts of Interest 45 The authors declare no competing financial interests. 46 47 48 Funding Sources 49 This work was supported by NIH grants R01DC016307 (F.I.) and R01MH116176 (Y.K.) 50 1 51 Abstract 52 In the mouse brain, olfactory information is transmitted to the olfactory cortex via olfactory bulb 53 (OB) projection neurons known as mitral and tufted cells. Although mitral and tufted cells share 54 many cellular characteristics, these cell types are distinct in their somata location and in their 55 axonal and dendritic projection patterns. Moreover, mitral cells consist of heterogeneous 56 subpopulations. We have previously shown that mitral cells generated at different embryonic 57 days differentially localize within the mitral cell layer and extend their lateral dendrites to 58 different sublayers of the external plexiform layer. Here, we examined the axonal projection 59 patterns from the subpopulations of OB projection neurons that are determined by the timing of 60 neurogenesis (neuronal birthdate) to understand the developmental origin of the diversity in 61 olfactory pathways. We separately labeled early- and late-generated OB projection neurons 62 using in utero electroporation performed at embryonic day 11 and 12, respectively, and 63 quantitatively analyzed their axonal projection patterns in the whole mouse brain using high- 64 resolution 3D imaging. In this study, we demonstrate that the axonal projection of late-generated 65 OB projection neurons is restricted to the anterior portion of the olfactory cortex while those of 66 the early-generated OB projection neurons innervate the entire olfactory cortex. Our results 67 suggest that the late-generated mitral cells do not extend their axons to the posterior regions of 68 the olfactory cortex. Therefore, the mitral cells having different birthdates differ, not only in cell 69 body location and dendritic projections within the OB, but also in their axonal projection pattern 70 to the olfactory cortex. 2 71 Significance Statement 72 The olfactory bulb contains long-range projection neurons with distinct connectivity to higher 73 order brain regions. Here, we examined how the birthdate of the olfactory bulb projection 74 neurons correlates to the generation of differential connectivity patterns. We used in utero 75 electroporation and high-resolution 3D imaging of the whole mouse brain, and determined the 76 topographically distinct axonal projection patterns of early- and late-generated olfactory bulb 77 projection neurons. Our results show that the timing of neurogenesis is a determining factor for 78 the innervation of olfactory bulb projection neurons and indicate that mitral cells having different 79 birthdates are the origins of distinct olfactory information pathways. Our study provides novel 80 insights into the formation of neuronal circuits processing multiple aspects of olfactory 81 information. 82 3 83 Introduction 84 The olfactory bulb (OB) is the first relay station for olfactory information in the vertebrate central 85 nervous system. Within the OB, projection neurons, mitral and tufted cells, receive input from 86 olfactory sensory neurons and transmit the olfactory information further to the olfactory cortex 87 consisting of several brain regions. Accumulating evidence suggests that distinct regions within 88 the olfactory cortex process different aspects of the olfactory information. For example, the 89 piriform cortex (PIR) is critical for odor discrimination, identification, and memory (Choi et al., 90 2011; Wilson and Sullivan, 2011; Bekkers and Suzuki, 2013; Blazing and Franks, 2020), the 91 anterior olfactory nucleus (AON) contributes to odor source detection (Kikuta et al., 2010; Liu et 92 al., 2020), the olfactory tubercle (OT) has close interaction with a reward system (Ikemoto, 93 2007; Wesson and Wilson, 2011; Gadziola et al., 2015; Yamaguchi, 2017; Zhang et al., 2017), 94 and the amygdala mediates the fear responses induced by predator odors (Root et al., 2014; 95 Isosaka et al., 2015; Kondoh et al., 2016). The segregation of the neural pathways controlling 96 these behavioral responses likely begins with diverse subpopulations of OB projection neurons 97 (Sosulski et al., 2011; Bear et al., 2016). 98 99 Historically, the major criterion to discriminate between mitral and tufted cells is somata location 100 within the OB. However, an increasing number of studies have reported differences in the 101 morphological and physiological properties between these two types of projection neurons in the 102 mammalian OB (Igarashi et al., 2012; Adam et al., 2014; Nagayama et al., 2014b; Cavarretta et 103 al., 2018). In particular, mitral and tufted cells project their axons to distinct regions in the 104 olfactory cortex. While a single mitral cell innervates almost the entire olfactory cortical areas, 105 tufted cells project axons only to the anterior portion of the olfactory cortex, including the OT and 106 AON (Nagayama et al., 2010; Igarashi et al., 2012; Hirata et al., 2019). This suggests that 107 different aspects of olfactory information are processed in parallel pathways originating from 108 mitral and tufted cells. In addition, recent studies have shown that mitral cells consist of 109 heterogeneous subpopulations with different cellular properties. Although mitral cells typically 110 extend their secondary dendrites in the deep sublayer of the external plexiform layer (EPL), 111 some mitral cells extend their secondary dendrites in the superficial sublayer of the EPL (Mori et 112 al., 1983; Orona et al., 1984; Mouradian and Scott, 1988). The diversity of intrinsic biophysical 113 properties among mitral cells, such as interspike interval, firing frequency, and the Ih sag 114 current, have also been reported (Nagayama et al., 2004; Padmanabhan and Urban, 2010; 115 Angelo et al., 2012; Igarashi et al., 2012). These differences in molecular and biophysical 116 properties may endow mitral cells with different odor response properties (Dhawale et al., 2010; 4 117 Kikuta et al., 2013). However, a critical question of whether different subsets of mitral cells 118 project axons to different regions in the olfactory cortex has yet to be answered. 119 120 In the developing mouse main OB, mitral cells are generated between embryonic days (E) 9 121 and 13, which is earlier than tufted cell birthdates (Hinds, 1968; Blanchart et al., 2006; Imamura 122 et al., 2011). We previously showed that early- and late-generated mitral cells were 123 preferentially localized at the dorsomedial and ventrolateral portion of the mitral cell layer (MCL), 124 respectively (Imamura et al., 2011). Furthermore, we separately labeled subsets of mitral cells 125 with different birthdates using the in utero electroporation method and revealed that early- and 126 late-generated mitral cells extend their lateral dendrites in the deep and superficial EPL, 127 respectively, (Imamura and Greer, 2015b). It has been speculated that neuronal birthdates may 128 also control the axonal projection patterns of OB projection neurons to the olfactory cortex 129 (Imamura et al., 2011; Hirata et al., 2019). These previous studies demonstrated that the OT 130 receives axonal inputs preferentially from tufted and late-generated mitral cells (Scott et al., 131 1980; Imamura et al., 2011), and segregated axonal projections are formed by early-generated 132 mitral cells and late-born external tufted cells (Hirata et al., 2019). Nevertheless, the axonal 133 projection of late-generated mitral cells to the olfactory cortex other than the OT, and differences 134 in axonal projection patterns between early- and late-generated mitral cells have not yet been 135 elucidated. In this study, we separately labeled the early- and late-generated OB projection 136 neurons using the in utero electroporation method and quantitatively analyzed axonal projection 137 patterns in the whole mouse brain using serial two-photon tomography (STPT) imaging.