A Protocol for Heterologous Expression and Functional Assay for Mouse Pheromone Receptors

Dey, Sandeepa; Zhan, Senmiao; Matsunami, Hiroaki

doi:10.1007/978-1-62703-619-1_9

Cited by 4 publications

(8 citation statements)

References 15 publications

(8 reference statements)

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“…However, other electrophysiological studies also failed to observe VSN adaptation when individual stimuli were delivered over a period of several hours 17 , 20 , 36 . Our in-vivo results show that pS6 immunoreactivity, employed as a marker of neuronal activity 30 , 39 , returns to background level in animals that are exposed to prolonged stimulation. However, electrophysiological adaptation occurs with a short latency after stimulus application, whereas the complete dephosphorylation of the S6 protein by a prolonged stimulation is a process that lasts several hours before it is fully accomplished.…”

Section: Discussionmentioning

confidence: 58%

In-vivo activation of vomeronasal neurons shows adaptive responses to pheromonal stimuli

Silvotti

Cavaliere

Belletti

et al. 2018

Sci Rep

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In most mammals, the vomeronasal system has a pivotal role in mediating socio-sexual behaviours. The vomeronasal organ senses pheromones through the activation of specific receptors. Pheromone binding to cognate receptors activates Ca-influx via the gating of a cation channel that generates membrane depolarisation. The ex-vivo activation of vomeronasal neurons (VSNs) by pheromonal stimuli has been largely investigated by electrophysiological and imaging techniques; however, few studies have been carried out to determine the physiological responses of VSNs, in-vivo. By tracking the phosphorylation of S6 ribosomal protein as a marker of neuronal activity, we show that S6 becomes phosphorylated (pS6) in mouse VSNs stimulated by intraspecific and heterospecific pheromonal cues. We observed that female scent induces pS6 immunoreactivity in the apical VSNs of male vomeronasal epithelium, whereas male cues stimulate S6 phosphorylation in both the basal and apical VSNs of females. We also show that this dimorphic pattern of pS6 immunoreactivity is reproduced when heterospecific stimuli are used. Moreover, we found that a consistent proportion of VSNs is activated by both heterospecific and intraspecific pheromones. Additionally, we have evidence of adaptive responses to S6 phosphorylation when stimulation with cues of the same and opposite sex and of different species is sustained.

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Section: Discussionmentioning

confidence: 58%

In-vivo activation of vomeronasal neurons shows adaptive responses to pheromonal stimuli

Silvotti

Cavaliere

Belletti

et al. 2018

Sci Rep

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“…An important step toward improving our understanding of mammalian BA chemosensation is to identify the peripheral receptors that endow animals with BA sensitivity. This continues to be a daunting task in the AOS, where limited tools have been available for receptor screening [although see (8,11,13)]. Rather than attempt to recreate heterologous expression systems (8,35,36), viral delivery methods (11), or transgenic mice for this purpose, we instead sought to develop a method that used primary VSNs in transgenic mice expressing GCaMP6f/s, drawing inspiration from (13).…”

Section: Fliccr-seq Supports the Identification Of Ba-sensitive Vrsmentioning

confidence: 99%

“…However, efforts to generate large-scale VR screening assays via heterologous expression has, for the most part, been unsuccessful [although see (8)]. Other efforts have explored VNO ligand-receptor interactions using various approaches (9)(10)(11)(12).…”

Section: Introductionmentioning

confidence: 99%

Physiology-forward identification of bile acid–sensitive vomeronasal receptors

et al. 2020

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“…This continues to be a daunting task in the AOS, where limited tools have been available for receptor screening (though see (Dey et al, 2013;Haga-Yamanaka et al, 2014;Lee et al, 2019)). Rather than attempt to recreate heterologous expression systems (Dey et al, 2013;Mainland and Matsunami, 2012;Saito et al, 2004), viral delivery methods (Lee et al, 2019), or transgenic mice for this purpose, we instead sought to develop a method that used primary VSNs in transgenic mice expressing GCaMP6f/s, drawing inspiration from (Haga-Yamanaka et al, 2014). The major drawback in taking this approach is that, assuming proportional expression of the ~300 known VRs and FPRs expressed by VSNs, less than 1% of the cells in the tissue express any given receptor (creating a "needle in a haystack" problem).…”

Section: Fliccr-seq Supports the Identification Of Ba-sensitive Vomermentioning

confidence: 99%

“…In theory, this feature would greatly simplify the process of unambiguously matching VSN ligands to their cognate receptors. However, efforts to generate large-scale VR screening assays via heterologous expression has, for the most part, been unsuccessful (though see (Dey et al, 2013)). Other efforts have explored VNO ligand-receptor interactions using various approaches (Haga-Yamanaka et al, 2015;Isogai et al, 2011;Lee et al, 2019;Stein et al, 2016).…”

mentioning

confidence: 99%

Physiology-forward identification of bile acid sensitive vomeronasal receptors

Wong

Cao

Zhang

et al. 2019

Preprint

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The mouse accessory olfactory system (AOS) supports social and reproductive behavior through the sensation of environmental chemosignals. A growing number of excreted steroids have been shown to be potent AOS cues, including bile acids (BAs) found in feces. As is still the case with most AOS ligands, the specific receptors used by vomeronasal sensory neurons (VSNs) to detect BAs remain unknown. To identify VSN BA receptors, we first performed a deep analysis of VSN BA tuning using volumetric GCaMP6f/s Ca 2+ imaging. These experiments revealed both broadly and narrowly tuned populations of BA-receptive VSNs with sub-micromolar sensitivities. We then developed a new physiology-forward approach for identifying AOS ligand-receptor interactions, which we call Fluorescence Live Imaging for Cell Capture and RNA-seq, or FLICCR-seq. FLICCR-seq analysis revealed 5 specific V1R-family receptors enriched in BA-sensitive VSNs. These studies introduce a powerful new approach for ligand-receptor matching and reveal biological mechanisms underlying mammalian BA chemosensation. Introduction:Chemosensory systems extract salient information from environmental cues that are critical for social and reproductive behaviors. In mice and other terrestrial mammals, the accessory olfactory system (AOS) guides innate behaviors such as territorial aggression and mating (Dulac and Torello, 2003;Keller et al., 2009). The initial detection of olfactory stimuli in the AOS is mediated by vomeronasal sensory neurons (VSNs), located in the vomeronasal organ (VNO), before these signals are routed to the accessory olfactory bulb and then behaviorally important regions including the medial amygdala and bed nucleus of the stria terminalis.Though the behavioral impacts of AOS chemosensation are manifold, our knowledge of the full complement of natural ligands for VSNs remains incomplete. Natural AOS ligands are found in the excretions of conspecific and heterospecific animals (e.g., urine, feces, tears, and saliva). These natural ligands include, but are not limited to, polar steroids, bile acids, major urinary proteins, and major histocompatibility complex peptide ligands (Chamero et al., 2007;Doyle et al., 2016;Leinders-Zufall et al., 2004;Nodari et al., 2008). As we learn more about the full natural complement of AOS ligands, new questions are arising about how these cues are detected by specific types of VSNs. Such knowledge is critical as we seek to understand how patterns of VSN activation by complex blends of environmental chemosignals cause changes in animal physiology and behavior.A major barrier preventing a deeper understanding of AOS function is a lack of ligandreceptor matches for AOS cues. VSNs generally express just one or two of ~300 unique vomeronasal receptors (VRs) or formyl peptide receptors (Dulac and Torello, 2003;Martini et al., 2001). The largely monoallelic expression of individual VRs by VSNs means that each VSN takes on the chemosensory sensitivity (or "tuning") of the dominantly expressed receptor. In theory, this feature wou...

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A Protocol for Heterologous Expression and Functional Assay for Mouse Pheromone Receptors

Cited by 4 publications

References 15 publications

In-vivo activation of vomeronasal neurons shows adaptive responses to pheromonal stimuli

In-vivo activation of vomeronasal neurons shows adaptive responses to pheromonal stimuli

Physiology-forward identification of bile acid–sensitive vomeronasal receptors

Physiology-forward identification of bile acid sensitive vomeronasal receptors

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