Embryonic and postnatal development of mouse olfactory tubercle

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Highlights

  • OT neurogenesis occurs between E11 and E15 in a lateral-to-medial maturation gradient

  • OT projection neurons generate in the vLGE and migrate through the "ventral migratory course"”

  • OT and striatal neurons share embryonic origin and differentiate acquiring similar molecular markers and morphologies

  • OT neuronal lineages have a layer restricted phenotype acquired early in the vLGE

  • OT dopaminergic innervation begins at E13 increasing gradually in an inside-out gradient

  • Myelination of OT layer III occurs postnatally between P7 and P14

Abstract

The olfactory tubercle (OT) is located in the ventral-medial region of the brain where it receives primary input from olfactory bulb (OB) projection neurons and processes olfactory behaviors related to motivation, hedonics of smell and sexual encounters. The OT is part of the dopamine reward system that shares characteristics with the striatum. Together with the nucleus accumbens, the OT has been referred to as the "ventral striatum". However, despite its functional importance little is known about the embryonic development of the OT and the phenotypic properties of the OT cells. Here, using thymidine analogs, we establish that mouse OT neurogenesis occurs predominantly between E11-E15 in a lateral-to-medial gradient. Then, using a piggyBac multicolor technique we characterized the migratory route of OT neuroblasts from their embryonic point of origin. Following neurogenesis in the ventral lateral ganglionic eminence (vLGE), neuroblasts destined for the OT followed a dorsal-ventral pathway we named "ventral migratory course" (VMC). Upon reaching the nascent OT, neurons established a prototypical laminar distribution that was determined, in part, by the progenitor cell of origin. A phenotypic analysis of OT neuroblasts using a single-color piggyBac technique, showed that OT shared the molecular specification of striatal neurons. In addition to primary afferent input from the OB, the OT also receives a robust dopaminergic input from ventral tegmentum (Ikemoto, 2007). We used tyrosine hydroxylase (TH) expression as a proxy for dopaminergic innervation and showed that TH onset occurs at E13 and progressively increased until postnatal stages following an ‘inside-out’ pattern. Postnatally, we established the myelination in the OT occurring between P7 and P14, as shown with CNPase staining, and we characterized the cellular phenotypes populating the OT by immunohistochemistry. Collectively, this work provides the first detailed analysis of the developmental and maturation processes occurring in mouse OT, and demonstrates the striatal nature of the OT as part of the ventral striatum (vST).

Introduction

The olfactory tubercle (OT) is located bilaterally in the ventral-medial brain and receives direct input from olfactory areas such as the piriform cortex (PC) and olfactory bulb (OB) (Barragan and Ferreyra-Moyano, 1988; Calleja, 1893; Nagayama et al., 2010; Narikiyo et al., 2014; Scott et al., 1980; Shipley and Adamek, 1984; Xiong and Wesson, 2016). The OT comprises 3 layers that have traditionally been classified as paleocortex: 1) the most superficial layer I or molecular/plexiform layer, constituted by an extraordinary number of neuronal fibers; 2) the intermediate layer II of densely packed cells or pyramidal/cortical layer; and 3) the internal cellular and fibrous layer III or polymorphic layer (Calleja, 1893; Ramon y Cajal, 1901). These layers include a variety of neuronal types (Millhouse and Heimer, 1984) organized in a characteristic rippled morphology caused by the presence of compact clusters of granule cells known as “islands of Calleja” (IC) (Adjei and Wesson, 2015; Calleja, 1893; De Marchis et al., 2004; Fallon et al., 1978; Giessel and Datta, 2014; Hsieh and Puche, 2013).

The OT is also widely connected to other brain areas such as the locus coeruleus, globus pallidus, nucleus of tractus solitarius, septum and the retina (Cooper et al., 1989; Fallon et al., 1978; Ruggiero et al., 1998; Solano-Flores et al., 1981; Switzer et al., 1982). This broad connectivity for both input and output functions renders the OT being a complicated structure involved in multiple distinct functions. The strong relationship of the OT with the striatal dopaminergic system, its shared cytoarchitectonic and histological properties with the striatum (ST), as well as the embryonic origin shared with the ST, has led to the suggestion that the OT be considered an extension of the ST, named collectively with the nucleus accumbens (NAc) and ventral pallidum (VP) the “ventral striatum” (vST) (Heimer, 1978; Heimer and Wilson, 1975; Millhouse, 1987; Wichterle et al., 2001; Xiong and Wesson, 2016). Consistent with this notion, the OT is implicated in the dopamine reward circuit (Ikemoto, 2007, Ikemoto, 2010), expressing elevated levels of dopamine receptors (Camps et al., 1990; Clement-Cormier et al., 1974; Versteeg et al., 1976) and having functions in the motivation and hedonics of odors (DiBenedictis et al., 2015; Fitzgerald et al., 2014; Gadziola and Wesson, 2016; Murata et al., 2015; Wesson and Wilson, 2010, Wesson and Wilson, 2011; Zelano et al., 2005). The hedonics of reward and the OT's strong innervation by dopamine may also implicate it in the rewarding effects of drugs of abuse (de Araujo et al., 2009; Haglund et al., 1979; Ikemoto, 2010; Kornetsky et al., 1991; Zhang et al., 2017a; Zhang et al., 2017b).

Comparatively little has been done on the developmental dynamics of the OT in mice. It seems clear that OT projection neurons and interneurons originate and migrate from the lateral (LGE) and medial (MGE) ganglionic eminences respectively, via tangential and radial migratory paths (Garcia-Moreno et al., 2008; Wichterle et al., 2001; Yun et al., 2003). However, although the OT is organized as a 3-layered structure, neurons in the OT are broadly considered subpopulations of striatal neurons and express similar markers such as choline acetyltransferase (ChAT), DARPP2, dopamine receptors (D1/D2/D3), Foxp2, glutamate receptors (GluRs and VGluts), 5-hydroxytryptamine (serotonin) receptor-4 (5-HT4), neurotensin (NT), neuropeptide-Y (NPY), RGS9, and somatostatin (SST) (Bischoff et al., 1997; Brunjes and Osterberg, 2015; Camps et al., 1990; Finley et al., 1978; Herzog et al., 2004; Kataoka et al., 1975; Murata et al., 2015; Schambra et al., 1989; Thomas et al., 1998; Wada et al., 1998; Waeber et al., 1996; Wichterle et al., 2001; Woodhams et al., 1985; Zahm, 1987).

To shed light on the development of the OT in mice and its similarities with ST, we report here an extensive analysis of the embryonic and postnatal development of the OT, including a characterization of the cellular phenotypes populating this structure and their specification during development. We first used thymidine analogs to study neurogenesis among the layers of the OT along the lateral-to-medial axis. We then used our piggyBac multicolor technique (Martin-Lopez et al., 2019) to characterize the migration of projection neurons targeted to the OT from their origin in the LGE. The multicolor strategy also enabled us to assess cell lineage and determine the role of progenitors in establishing cell fate. A similar strategy, but using a single-fluorophore piggyBac plasmid, was used to guarantee the specification process of OT neuroblasts while migrating and at mature stages, demonstrating they shared molecular phenotypes with ST neurons. We also used tyrosine hydroxylase (TH) expression as a proxy to determine the timeline of dopaminergic innervation of the OT, and developmental patterns of myelination using CNPase staining. Finally, we characterized the cellular phenotypes occupying the mature OT by immunohistochemistry, using a complete array of markers detecting neuron and glial cells.

Section snippets

Animals

CD1 mice, both male and female, were used for the experiments and obtained from Charles River Laboratories (Wilmington, MA). For experiments at embryonic stages, we considered embryonic age 0 (E0) the day the vaginal plug was found. All animals were housed in a standard 12-h light cycle with ad libitum access to standard chow. All procedures were approved by Yale University Animal Care and Use Committee.

Injections of thymidine analogs and processing of tissue

For neurogenesis analysis, one group of pregnant females was injected intraperitoneally (IP)

Neurogenesis of the olfactory tubercle

Neurogenesis in the mouse OT was originally addressed using incorporation of 3H-thymidine (Creps, 1974), which showed an ‘inside-out’ developmental gradient beginning at ~E10. Bayer (1985) suggested further a lateral-to-medial axis of maturation in the rat OT, but the issue of a lateral to medial gradient was not examined in mice. Similarly, the migratory path(s) of neuroblasts targeted to the OT or aspects of their lineage were not reported. Here, we first examined OT neurogenesis using BrdU

Discussion

The OT has been studied largely as part of the dopaminergic reward system of the vST, implicating this structure in multiple odor-related behaviors including feeding, sexual behaviors, attention-sniffing, odor preference, motivation and hedonics of smell (DiBenedictis et al., 2015; Fitzgerald et al., 2014; Gadziola et al., 2015; Koob et al., 1978; Menna-Barreto et al., 1983; Murata et al., 2015; Perkins et al., 1980; Wesson and Wilson, 2011; Zelano et al., 2005; Zhang et al., 2017a). As a

Acknowledgments

We thank Jaime Grutzendler for the CNPase antibody, and the very helpful discussions with all members of the Greer Lab. This work was supported in part by NIH DC012441, DC015438 and DC013791.

Conflict of interests

None declared.

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