Mouse Accessory Olfactory System Research Articles

Paradoxically Sparse Chemosensory Tuning in Broadly Integrating External Granule Cells in the Mouse Accessory Olfactory Bulb.

The accessory olfactory bulb (AOB), the first neural circuit in the mouse accessory olfactory system, is critical for interpreting social chemosignals. Despite its importance, AOB information processing is poorly understood compared with the main olfactory bulb (MOB). Here, we sought to fill gaps in the understanding of AOB interneuron function. We used 2-photon GCaMP6f Ca2+ imaging in an ex vivo preparation to study chemosensory tuning in AOB external granule cells (EGCs), interneurons hypothesized to broadly inhibit activity in excitatory mitral cells (MCs). In ex vivo preparations from mice of both sexes, we measured MC and EGC tuning to natural chemosignal blends and monomolecular ligands, finding that EGC tuning was sparser, not broader, than upstream MCs. Simultaneous electrophysiological recording and Ca2+ imaging showed no differences in GCaMP6f-to-spiking relationships in these cell types during simulated sensory stimulation, suggesting that measured EGC sparseness was not due to cell type-dependent variability in GCaMP6f performance. Ex vivo patch-clamp recordings revealed that EGC subthreshold responsivity was far broader than indicated by GCaMP6f Ca2+ imaging, and that monomolecular ligands rarely elicited EGC spiking. These results indicate that EGCs are selectively engaged by chemosensory blends, suggesting different roles for EGCs than analogous interneurons in the MOB.SIGNIFICANCE STATEMENT The mouse accessory olfactory system (AOS) interprets social chemosignals, but we poorly understand AOS information processing. Here, we investigate the functional properties of external granule cells (EGCs), a major class of interneurons in the accessory olfactory bulb (AOB). We hypothesized that EGCs, which are densely innervated by excitatory mitral cells (MCs), would show broad chemosensory tuning, suggesting a role in divisive normalization. Using ex vivo GCaMP6f imaging, we found that EGCs were instead more sparsely tuned than MCs. This was not due to weaker GCaMP6f signaling in EGCs than in MCs. Instead, we found that many MC-activating chemosignals caused only subthreshold EGC responses. This indicates a different role for AOB EGCs compared with analogous cells in the main olfactory bulb.

Physiology-forward identification of bile acid-sensitive vomeronasal receptors.

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 Ca2+ imaging. These experiments revealed multiple populations of BA-receptive VSNs with submicromolar 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 sequencing, or FLICCR-seq. FLICCR-seq analysis revealed five 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.

Bacterial MgrB peptide activates chemoreceptor Fpr3 in mouse accessory olfactory system and drives avoidance behaviour

Innate immune chemoreceptors of the formyl peptide receptor (Fpr) family are expressed by vomeronasal sensory neurons (VSNs) in the accessory olfactory system. Their biological function and coding mechanisms remain unknown. We show that mouse Fpr3 (Fpr-rs1) recognizes the core peptide motif f-MKKFRW that is predominantly present in the signal sequence of the bacterial protein MgrB, a highly conserved regulator of virulence and antibiotic resistance in Enterobacteriaceae. MgrB peptide can be produced and secreted by bacteria, and is selectively recognized by a subset of VSNs. Exposure to the peptide also stimulates VSNs in freely behaving mice and drives innate avoidance. Our data shows that Fpr3 is required for neuronal detection and avoidance of peptides derived from a conserved master virulence regulator of enteric bacteria.

Experience-Dependent Plasticity in Accessory Olfactory Bulb Interneurons following Male-Male Social Interaction.

Chemosensory information processing in the mouse accessory olfactory system guides the expression of social behavior. After salient chemosensory encounters, the accessory olfactory bulb (AOB) experiences changes in the balance of excitation and inhibition at reciprocal synapses between mitral cells (MCs) and local interneurons. The mechanisms underlying these changes remain controversial. Moreover, it remains unclear whether MC-interneuron plasticity is unique to specific behaviors, such as mating, or whether it is a more general feature of the AOB circuit. Here, we describe targeted electrophysiological studies of AOB inhibitory internal granule cells (IGCs), many of which upregulate the immediate-early gene Arc after male-male social experience. Following the resident-intruder paradigm, Arc-expressing IGCs in acute AOB slices from resident males displayed stronger excitation than nonexpressing neighbors when sensory inputs were stimulated. The increased excitability of Arc-expressing IGCs was not correlated with changes in the strength or number of excitatory synapses with MCs but was instead associated with increased intrinsic excitability and decreased HCN channel-mediated IH currents. Consistent with increased inhibition by IGCs, MCs responded to sensory input stimulation with decreased depolarization and spiking following resident-intruder encounters. These results reveal that nonmating behaviors drive AOB inhibitory plasticity and indicate that increased MC inhibition involves intrinsic excitability changes in Arc-expressing interneurons.SIGNIFICANCE STATEMENT The accessory olfactory bulb (AOB) is a site of experience-dependent plasticity between excitatory mitral cells (MCs) and inhibitory internal granule cells (IGCs), but the physiological mechanisms and behavioral conditions driving this plasticity remain unclear. Here, we report studies of AOB neuronal plasticity following male-male social chemosensory encounters. We show that the plasticity-associated immediate-early gene Arc is selectively expressed in IGCs from resident males following the resident-intruder assay. After behavior, Arc-expressing IGCs are more strongly excited by sensory input stimulation and MC activation is suppressed. Arc-expressing IGCs do not show increased excitatory synaptic drive but instead show increased intrinsic excitability. These data indicate that MC-IGC plasticity is induced after male-male social chemosensory encounters, resulting in enhanced MC suppression by Arc-expressing IGCs.

Faecal bile acids are natural ligands of the mouse accessory olfactory system.

The accessory olfactory system (AOS) guides behaviours that are important for survival and reproduction, but understanding of AOS function is limited by a lack of identified natural ligands. Here we report that mouse faeces are a robust source of AOS chemosignals and identify bile acids as a class of natural AOS ligands. Single-unit electrophysiological recordings from accessory olfactory bulb neurons in ex vivo preparations show that AOS neurons are strongly and selectively activated by peripheral stimulation with mouse faecal extracts. Faecal extracts contain several unconjugated bile acids that cause concentration-dependent neuronal activity in the AOS. Many AOS neurons respond selectively to bile acids that are variably excreted in male and female mouse faeces, and others respond to bile acids absent in mouse faeces. These results identify faeces as a natural source of AOS information, and suggest that bile acids may be mammalian pheromones and kairomones.

Ex vivo preparations of the intact vomeronasal organ and accessory olfactory bulb.

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (<1 mm(3)) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Robust Encoding of Stimulus Identity and Concentration in the Accessory Olfactory System

Sensory systems represent stimulus identity and intensity, but in the neural periphery these two variables are typically intertwined. Moreover, stable detection may be complicated by environmental uncertainty; stimulus properties can differ over time and circumstance in ways that are not necessarily biologically relevant. We explored these issues in the context of the mouse accessory olfactory system, which specializes in detection of chemical social cues and infers myriad aspects of the identity and physiological state of conspecifics from complex mixtures, such as urine. Using mixtures of sulfated steroids, key constituents of urine, we found that spiking responses of individual vomeronasal sensory neurons encode both individual compounds and mixtures in a manner consistent with a simple model of receptor-ligand interactions. Although typical neurons did not accurately encode concentration over a large dynamic range, from population activity it was possible to reliably estimate the log-concentration of pure compounds over several orders of magnitude. For binary mixtures, simple models failed to accurately segment the individual components, largely because of the prevalence of neurons responsive to both components. By accounting for such overlaps during model tuning, we show that, from neuronal firing, one can accurately estimate log-concentration of both components, even when tested across widely varying concentrations. With this foundation, the difference of logarithms, log A - log B = log A/B, provides a natural mechanism to accurately estimate concentration ratios. Thus, we show that a biophysically plausible circuit model can reconstruct concentration ratios from observed neuronal firing, representing a powerful mechanism to separate stimulus identity from absolute concentration.

Representation and transformation of sensory information in the mouse accessory olfactory system

In mice, nonvolatile social cues are detected and analyzed by the accessory olfactory system (AOS). Here we provide a first view of information processing in the AOS with respect to individual chemical cues. 12 sulfated steroids, recently-discovered mouse AOS ligands, caused widespread activity among vomeronasal sensory neurons (VSNs), yet VSN responses clustered into a small number of repeated functional patterns or processing streams. Downstream neurons in the accessory olfactory bulb (AOB) responded to these ligands with enhanced signal/noise compared to VSNs. Whereas the dendritic connectivity of AOB mitral cells suggests the capacity for broad integration, most sulfated steroid responses were well-modeled by linear excitatory drive from just one VSN processing stream. However, a significant minority demonstrated multi-stream integration. Most VSN excitation patterns were also observed in the AOB, but excitation by estradiol sulfate processing streams was rare, suggesting AOB circuit organization is specific to the biological relevance of sensed cues.

Removal of the superior cervical ganglia fails to block Fos induction in the accessory olfactory system of male mice after exposure to female odors

When exposed to female odors, testosterone-primed male mice show a robust expression of immediate early genes in the vomeronasal organ (VNO) and associated accessory olfactory structures. We asked whether the superior cervical ganglia (SCG), which provide autonomic inputs to the VNO, are required for odor induction of Fos. Gonadally intact male mice received sham, unilateral, or bilateral SCG lesions and were exposed to odors from estrous females. In comparison to clean bedding, female odors significantly increased neuronal Fos immunoreactivity in sites throughout the VNO projection pathway, but these responses were not reliably modified by SCG removal. Thus, noradrenergic inputs to the VNO, which regulate a pumping mechanism thought to facilitate entrance of chemosignals into the VNO lumen, are not required for odors to induce Fos in the mouse accessory olfactory system.

Sex Difference and Steroid Modulation of Pheromone-Induced Immediate Early Genes in the Two Zones of the Mouse Accessory Olfactory System

Two anatomically and neurochemically distinct zones within the vomeronasal organ (VNO) and accessory olfactory bulb (AOB) have been identified that are responsible for the detection of pheromones. Using markers to distinguish between apical and basal neurons of the VNO neuroepithelium and rostral versus caudal AOB glomeruli, we examined immediate early gene immunoreactivity (IEG-IR) in gonadectomized, steroid-treated mice in response to pheromones of male and female conspecifics. After exposure of estradiol-treated females to soiled male bedding, more VNO neurons in the basal than the apical layer exhibited IEG-IR compared with VNO neurons of estradiol-treated males. Conversely, whereas soiled female bedding failed to induce IEG-IR in VNO neurons of estradiol-treated males or females, both apical and basal neurons were activated in testosterone-treated males. Male and female pheromones also activated mitral and granule cells in the AOBs of all subjects, but responses to different pheromones were distributed across the boundary of the rostral and caudal regions. These data show that differences in the response of males and females to the same pheromonal stimulus are found in the sensory neurons of the VNO. We propose that centrifugal, noradrenergic inputs to VNO neurons, which may differ in the two sexes and respond differently to adult sex steroids, modulate sensitivity to pheromonal stimulation.

Olfaction in utero: Behavioral studies of the mouse fetus

On the day before birth, mouse fetuses were tested for their behavioral responses to iso-amyl acetate and iso-valeric acid delivered into the nasal cavity in liquid phase. Pilot studies established that most subjects responded to most concentrations used with an increase in total behaviors and number of different behaviors displayed. When iso-amyl acetate was systematically delivered at three concentrations (10 −3 M, 10 −4 M and 10 −5 M) subjects showed significant responses to each. In contrast, responses were not evident when saline was the stimulus. The magnitude of the average behavioral response did not decline with decreasing concentration of odorant. In a second experiment, the ability of the fetus to discriminate between iso-amyl acetate and iso-valeric acid was studied. Results establish that the fetuses responded differently to the two odorants. Given the immaturity of the mouse's accessory olfactory system before birth and the observed responses to concentrations of odorants below the threshold of the trigeminal system, the results suggest that the mouse fetus has a functionally competent main olfactory system including the ability to discriminate between purified odorants. The results are discussed in terms of current models of chemosensory ontogeny.