Reducing local synthesis of estrogen in the tubular striatum promotes attraction to same-sex odors in female mice

Highlights

• Sex hormones such as 17β-estradiol influence odor-directed behavior.

• The tubular striatum (TuS) is important for odor-guided motivated behavior.

• We examined odor-evoked sniffing following TuS aromatase inhibition via letrozole.

• TuS aromatase inhibition in females promoted male-typical attraction to female odors.

• Males displayed sustained attraction to female odors regardless of treatment.

Abstract

Brain-derived 17β-estradiol (E2) confers rapid effects on neural activity. The tubular striatum (TuS, also called the olfactory tubercle) is both capable of local E2 synthesis due to its abundant expression of aromatase and is a critical locus for odor-guided motivated behavior and odor hedonics. TuS neurons also contain mRNA for estrogen receptors α, β, and the G protein-coupled estrogen receptor. We demonstrate here that mRNA for estrogen receptors appears to be expressed upon TuS dopamine 1 receptor-expressing neurons, suggesting that E2 may play a neuromodulatory role in circuits which are important for motivated behavior. Therefore, we reasoned that E2 in the TuS may influence attraction to urinary odors which are highly attractive. Using whole-body plethysmography, we examined odor-evoked high-frequency sniffing as a measure of odor attaction. Bilateral infusion of the aromatase inhibitor letrozole into the TuS of gonadectomized female adult mice induced a resistance to habituation over successive trials in their investigatory sniffing for female mouse urinary odors, indicative of an enhanced attraction. All males displayed resistance to habituation for female urinary odors, indicative of enhanced attraction that is independent from E2 manipulation. Letrozole's effects were not due to group differences in basal respiration, nor changes in the ability to detect or discriminate between odors (both monomolecular odorants and urinary odors). Therefore, de novo E2 synthesis in the TuS impacts females' but not males' attraction to female urinary odors, suggesting a sex-specific influence of E2 in odor hedonics.

Introduction

Estrogens broadly influence behavior beyond their well-established roles in regulating sexual development. In particular, 17β-estradiol (E2) is important for the regulation of mood, learning and memory, and sensory-guided behavior (e.g., (Walf and Frye, 2006; Tuscher et al., 2016; Kunkhyen et al., 2018; Vahaba and Remage-Healey, 2018; Koss and Frick, 2019; Lu et al., 2019)). Recent evidence points to a particulary impactful role of E2 that is synthesized de novo in the brain. Aromatase, the enzyme responsible for E2 synthesis, is expressed in numerous brain regions and across species, in both sexes (Naftolin et al., 1971; MacLusky and Naftolin, 1981; Roselli and Resko, 1989; Vockel et al., 1990). Compared to ovarian-derived E2, local E2 synthesis may influence neuroplasticity on a more rapid timescale (within minutes) via actions on membrane-bound estrogen receptors (Razandi et al., 2003; Thomas et al., 2005; Meitzen and Mermelstein, 2011). Indeed, even after gonadectomy (GDX) to deplete peripheral sex steroid hormones, E2 levels remain high in brain regions containing aromatase (Li and Gibbs, 2019), suggesting a unique role for brain-derived E2 compared to ovarian-derived E2.

GDX and aromatase knockout mice exhibit sex-dependent changes in perception and behaviors toward social cues, specifically odors. For instance, GDX and aromatase knockout mice have deficits in odor discrimination for both ethologically-relevant odors and nonethologically-relevant odors which are not innately attractive, such as monomolecular odorants (Wesson et al., 2006; Sorwell et al., 2008; Xiao et al., 2015; Kunkhyen et al., 2018). This indicates that systemic E2 may influence ethologically critical odor-guided behaviors. However, these studies do not investigate the specific brain regions in which local E2 synthesis would support these influences of E2 on odor-guided behaviors.

One candidate brain region for investigating neurosteroids' effects on odor-guided behavior is the tubular striatum (TuS, (Wesson, 2020), also known as the olfactory tubercle). The TuS is a primary target of olfactory bulb sensory input and is active during tasks involving odor-guided attention and investigatory sniffing (Zelano et al., 2005; Payton et al., 2012; Carlson et al., 2018; Oettl et al., 2020). There is ample expression of aromatase, estrogen receptors alpha (ERα) and beta (ERβ), and the G protein-coupled estrogen receptor (GPER) in the TuS (Rainbow et al., 1982; Shughrue et al., 1997; Brailoiu et al., 2007; Roselli and Stormshak, 2012; Stanić et al., 2014), and the activity of TuS neurons appears sensitive to changes in estrogen levels (Cartas-Heredia et al., 1978). TuS neurons are primarily GABAergic medium spiny neurons (MSNs) expressing the dopamine D1 type or D2 type receptors (Murata et al., 2015; Zhang et al., 2017b; White et al., 2019)– neurons shown to have opposing roles in motivated behavior (Lobo and Nestler, 2011; Murata et al., 2019). TuS D1 MSNs are activated by odors conditioned to predict rewards, and activation of D1 MSNs promotes approach behaviors (Murata et al., 2019; Gadziola et al., 2020). The TuS, along with the neighboring nucleus accumbens, comprise the ventral striatum, which undergoes significant sex- and hormone-dependent alterations in dendritic spine density, input excitability, and intrinsic properties of its MSNs (Forlano and Woolley, 2009; Wissman et al., 2012; Cao et al., 2018; Proaño et al., 2018; Krentzel et al., 2019). TuS neurons appear necessary for driving attraction to intrinsically-rewarding odors (Agustín-Pavón et al., 2014; FitzGerald et al., 2014; DiBenedictis et al., 2015; Zhang et al., 2017a) in a manner that is dependent on midbrain dopaminergic input (DiBenedictis et al., 2014; Zhang et al., 2017a). Together, these results support a model whereby hormonal modulation of TuS neurons may influence odor-guided behaviors including those which are specific to sex, but it is unknown whether TuS-derived estrogens influence odor attraction and in which TuS neurons estrogen receptors are expressed.

In this study, we first examined the expression profile of estrogen receptors to determine whether there is neuronal subtype-specific expression in the TuS, since actions of E2 may differ depending upon which TuS MSNs they are expressed. We next tested the specific hypothesis that TuS-derived E2 rapidly modulates olfactory attraction in a sex-dependent manner. To do this, we examined the impact of aromatase inhibition in the TuS on attraction toward ethologically-salient and non-ethologically-salient odors using whole-body plethysmography. This approach allowed for a sensitive and quantitative measure of odor attraction by examining displays of sniffing frequency when mice were either injected with letrozole or, on separate days, vehicle into their TuS.