In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ.
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ABSTRACT:
SUBMITTER: Ackels T
PROVIDER: S-EPMC5091996 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The mouse vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Based on largely monogenic expression of either type 1 or 2 vomeronasal receptors (V1Rs/V2Rs) or members of the formyl peptide receptor (FPR) family, the vomeronasal sensory epithelium harbors at least three neuronal subpopulations. While various neurophysiological properties of both V1R- and V2R-expressing neurons have been described using genetically engineered mouse models, the basic biophysical characteristics of the more recently identified FPR-expressing vomeronasal neurons have not been studied. Here, we employ a transgenic mouse strain that coexpresses an enhanced variant of yellow fluorescent protein together with FPR-rs3 allowing to identify and analyze FPR-rs3-expressing neurons in acute VNO tissue slices. Single neuron electrophysiological recordings allow comparative characterization of the biophysical properties inherent to a prototypical member of the FPR-expressing subpopulation of VNO neurons. In this study, we provide an in-depth analysis of both passive and active membrane properties, including detailed characterization of several types of voltage-activated conductances and action potential discharge patterns, in fluorescently labeled vs. unmarked vomeronasal neurons. Our results reveal striking similarities in the basic (electro) physiological architecture of both transgene-expressing and non-expressing neurons, confirming the suitability of this genetically engineered mouse model for future studies addressing more specialized issues in vomeronasal FPR neurobiology.
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Project description:The mammalian vomeronasal organ detects complex chemical signals that convey information about gender, strain, and the social and reproductive status of an individual. How these signals are encoded is poorly understood. We developed transgenic mice expressing the calcium indicator G-CaMP2 and analyzed population responses of vomeronasal neurons to urine from individual animals. A substantial portion of cells was activated by either male or female urine, but only a small population of cells responded exclusively to gender-specific cues shared across strains and individuals. Female cues activated more cells and were subject to more complex hormonal regulations than male cues. In contrast to gender, strain and individual information was encoded by the combinatorial activation of neurons such that urine from different individuals activated distinctive cell populations.
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Project description:The physiology and pathophysiology of the exocrine pancreas are in close connection to changes in intra-cellular Ca2+ concentration. Most of our knowledge is based on in vitro experiments on acinar cells or acini enzymatically isolated from their surroundings, which can alter their structure, physiology, and limit our understanding. Due to these limitations, the acute pancreas tissue slice technique was introduced almost two decades ago as a complementary approach to assess the morphology and physiology of both the endocrine and exocrine pancreas in a more conserved in situ setting. In this study, we extend previous work to functional multicellular calcium imaging on acinar cells in tissue slices. The viability and morphological characteristics of acinar cells within the tissue slice were assessed using the LIVE/DEAD assay, transmission electron microscopy, and immunofluorescence imaging. The main aim of our study was to characterize the responses of acinar cells to stimulation with acetylcholine and compare them with responses to cerulein in pancreatic tissue slices, with special emphasis on inter-cellular and inter-acinar heterogeneity and coupling. To this end, calcium imaging was performed employing confocal microscopy during stimulation with a wide range of acetylcholine concentrations and selected concentrations of cerulein. We show that various calcium oscillation parameters depend monotonically on the stimulus concentration and that the activity is rather well synchronized within acini, but not between acini. The acute pancreas tissue slice represents a viable and reliable experimental approach for the evaluation of both intra- and inter-cellular signaling characteristics of acinar cell calcium dynamics. It can be utilized to assess many cells simultaneously with a high spatiotemporal resolution, thus providing an efficient and high-yield platform for future studies of normal acinar cell biology, pathophysiology, and screening pharmacological substances.
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Project description:In mammalian species, detection of pheromone cues by the vomeronasal organ (VNO) at different concentrations can elicit distinct behavioral responses and endocrine changes. It is not well understood how concentration-dependent activation of the VNO impacts innate behaviors. In this study, we find that, when mice investigate the urogenital areas of a conspecific animal, the urinary pheromones can reach the VNO at a concentration of approximately 1% of that in urine. At this level, urinary pheromones elicit responses from a subset of cells that are tuned to sex-specific cues and provide unambiguous identification of the sex and strain of animals. In contrast, low concentrations of urine do not activate these cells. Strikingly, we find a population of neurons that is only activated by low concentrations of urine. The properties of these neurons are not found in neurons responding to putative single-compound pheromones. Additional analyses show that these neurons are masked by high-concentration pheromones. Thus, an antagonistic interaction in natural pheromones results in the activation of distinct populations of cells at different concentrations. The differential activation is likely to trigger different downstream circuitry and underlies the concentration-dependent pheromone perception.
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Project description:In endocrine cells within islets of Langerhans calcium ions couple cell stimulation to hormone secretion. Since the advent of modern fluorimetry, numerous in vitro studies employing primarily isolated mouse islets have investigated the effects of various secretagogues on cytoplasmic calcium, predominantly in insulin-secreting beta cells. Due to technical limitations, insights of these studies are inherently limited to a rather small subpopulation of outermost cells. The results also seem to depend on various factors, like culture conditions and duration, and are not always easily reconcilable with findings in vivo. The main controversies regard the types of calcium oscillations, presence of calcium waves, and the level of synchronized activity. Here, we set out to combine the in situ acute mouse pancreas tissue slice preparation with noninvasive fluorescent calcium labeling and subsequent confocal laser scanning microscopy to shed new light on the existing controversies utilizing an innovative approach enabling the characterization of responses in many cells from all layers of islets. Our experiments reproducibly showed stable fast calcium oscillations on a sustained plateau rather than slow oscillations as the predominant type of response in acute tissue slices, and that calcium waves are the mechanistic substrate for synchronization of oscillations. We also found indirect evidence that even a large amplitude calcium signal was not sufficient and that metabolic activation was necessary to ensure cell synchronization upon stimulation with glucose. Our novel method helped resolve existing controversies and showed the potential to help answer important physiological questions, making it one of the methods of choice for the foreseeable future.
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Project description:BACKGROUND:The chemosensory system plays an important role in orchestrating sexual behaviors in mammals. Pheromones trigger sexually dimorphic behaviors and different mouse strains exhibit differential responses to pheromone stimuli. It has been speculated that differential gene expression in the sensory organs that detect pheromones may underlie sexually-dimorphic and strain-specific responses to pheromone cues. RESULTS:We have performed transcriptome analyses of the mouse vomeronasal organ, a sensory organ recognizing pheromones and interspecies cues. We find little evidence of sexual dimorphism in gene expression except for Xist, an essential gene for X-linked gene inactivation. Variations in gene expression are found mainly among strains, with genes from immune response and chemosensory receptor classes dominating the list. Differentially expressed genes are concentrated in genomic hotspots enriched in these families of genes. Some chemosensory receptors show exclusive patterns of expression in different strains. We find high levels of single nucleotide polymorphism in chemosensory receptor pseudogenes, some of which lead to functionalized receptors. Moreover, we identify a number of differentially expressed long noncoding RNA species showing strong correlation or anti-correlation with chemoreceptor genes. CONCLUSIONS:Our analyses provide little evidence supporting sexually dimorphic gene expression in the vomeronasal organ that may underlie dimorphic pheromone responses. In contrast, we find pronounced variations in the expression of immune response related genes, vomeronasal and G-protein coupled receptor genes among different mouse strains. These findings raised the possibility that diverse strains of mouse perceive pheromone cues differently and behavioral difference among strains in response to pheromone may first arise from differential detection of pheromones. On the other hand, sexually dimorphic responses to pheromones more likely originate from dimorphic neural circuits in the brain than from differential detection. Moreover, noncoding RNA may offer a potential regulatory mechanism controlling the differential expression patterns.
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Project description:The vomeronasal system consists of a peripheral organ and the connected central neuronal networks. The central connections are sexually dimorphic in rodents, and in some species, parameters of the vomeronasal organ (VNO) vary with sex, hormonal exposure, body size and seasonality. The VNO of the dasyurid marsupial mouse, Antechinus subtropicus is presumed to be functional. The unusual life history (male semelparity) is marked by distinct seasonality with differences in hormonal environments both between males and females, and in males at different time points. Body size parameters (e.g., length, weight) display sexual dimorphism and, in males, a pronounced weight gain before breeding is followed by a rapid decline during the single, short reproductive season. VNO morphometry was investigated in male and female A. subtropicus to identify possible life cycle associated activity. The overall length of the VNO is positively correlated with the size of the animal. The amount of sensory epithelium exhibits a negative correlation, decreasing with increasing size of the animal. The effects of sex and breeding condition are not obvious, although they do suggest that sensory vomeronasal epithelium mass declines in the breeding period. The VNO may be more important in A. subtropicus before breeding when it may participate in synchronising reproduction and in the development of the male stress response. J. Morphol. 277:1517-1530, 2016. © 2016 Wiley Periodicals, Inc.
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Project description:Here, we have developed an open multi-organ communication device that facilitates cellular and molecular communication between ex vivo organ slices. Measuring communication between organs is vital for understanding the mechanisms of health regulation yet remains difficult with current technology. Communication between organs along the gut-brain-immune axis is a key regulator of gut homeostasis. As a novel application of the device, we have used tissue slices from the Peyer's patch (PP) and mesenteric lymph node (MLN) due to their importance in gut immunity; however, any organ slices could be used here. The device was designed and fabricated using a combination of 3D printed molds for polydimethylsiloxane (PDMS) soft lithography, PDMS membranes, and track-etch porous membranes. To validate cellular and protein transfer between organs on-chip, we used fluorescence microscopy to quantitate movement of fluorescent proteins and cells from the PP to the MLN, replicating the initial response to immune stimuli in the gut. IFN-γ secretion during perfusion from a naïve vs. inflamed PP to a healthy MLN was quantitated to demonstrate soluble signaling molecules are moving on-chip. Finally, transient catecholamine release was measured during perfusion from PP to MLN using fast-scan cyclic voltammetry at carbon-fiber microelectrodes to demonstrate a novel application of the device for real-time sensing during communication. Overall, we show an open-well multi-organ device capable of facilitating transfer of soluble factors and cells with the added benefit of being available for external analysis techniques like electrochemical sensing which will advance abilities to probe communication in real-time across multiple organs ex vivo.
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Project description:Controlling stimulus access to sensory organs allows animals to optimize sensory reception and prevent damage. The vomeronasal organ (VNO) detects pheromones and other semiochemicals to regulate innate social and sexual behaviors. This semiochemical detection generally requires the VNO to draw in chemical fluids, such as bodily secretions, which are complex in composition and can be contaminated. Little is known about whether and how chemical constituents are monitored to regulate the fluid access to the VNO. Using transgenic mice and immunolabeling, we found that solitary chemosensory cells (SCCs) reside densely at the entrance duct of the VNO. In this region, most of the intraepithelial trigeminal fibers innervate the SCCs, indicating that SCCs relay sensory information onto the trigeminal fibers. These SCCs express transient receptor potential channel M5 (TRPM5) and the phospholipase C (PLC) beta2 signaling pathway. Additionally, the SCCs express choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) for synthesizing and packaging acetylcholine, a potential transmitter. In intracellular Ca2+ imaging, the SCCs responded to various chemical stimuli including high concentrations of odorants and bitter compounds. The responses were suppressed significantly by a PLC inhibitor, suggesting involvement of the PLC pathway. Further, we developed a quantitative dye assay to show that the amount of stimulus fluid that entered the VNOs of behaving mice is inversely correlated to the concentration of odorous and bitter substances in the fluid. Genetic knockout and pharmacological inhibition of TRPM5 resulted in larger amounts of bitter compounds entering the VNOs. Our data uncovered that chemoreception of fluid constituents regulates chemical access to the VNO and plays an important role in limiting the access of non-specific irritating and harmful substances. Our results also provide new insight into the emerging role of SCCs in chemoreception and regulation of physiological actions.
| S-EPMC2912856 | biostudies-literature
Project description:The vomeronasal organ (VNO) is essential for intraspecies communication in many terrestrial vertebrates. The ionic mechanisms of VNO activation remain unclear. We found that the calcium-activated potassium channel SK3 and the G protein-activated potassium channel GIRK are part of an independent pathway for VNO activation. In slice preparations, the potassium channels attenuated inward currents carried by TRPC2 and calcium-activated chloride channels (CACCs). In intact tissue preparations, paradoxically, the potassium channels enhanced urine-evoked inward currents. This discrepancy resulted from the loss of a high concentration of lumenal potassium, which enabled the influx of potassium ions to depolarize the VNO neurons in vivo. Both Sk3 (also known as Kcnn3) and Girk1 (also known as Kcnj3) homozygous null mice showed deficits in mating and aggressive behaviors, and the deficiencies in Sk3(-/-) mice were exacerbated by Trpc2 knockout. Our results suggest that VNO activation is mediated by TRPC2, CACCs and two potassium channels, all of which contributed to the in vivo depolarization of VNO neurons.
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