Number Of Subpopulations Research Articles

Intraindividual long term stability and response corridors of cytokines in healthy volunteers detected by a standardized whole-blood culture system for bed-side application

BackgroundThe variation of immune cell activities over time is an immanent property of the human immune system, as can be measured by the stimulated secretion of cytokines in cell cultures. However, inter-individual variability is considerably higher. Especially the latter is the major reason why it has not been possible to establish international standard values for cytokines as was possible for other parameters, such as leukocyte sub-population numbers. In this trial, a highly standardized whole-blood culture model (TrueCulture®), developed to characterise drug effects on cells of the human immune system in clinical trials, was used to analyse cytokine patterns in the blood samples of 12 healthy subjects over a period of one month.MethodsAfter an overnight fast, 12 healthy subjects donated blood three times a week on three consecutive days over a period of 4 weeks. TruCulture® blood collection and whole-blood culture systems were used to measure whole-blood leukocyte stimulation. The levels of IL-2, IL-5, IL-13, IL-6, IL-8, IL-10, IFNγ, and MCP-1 in the culture supernatants were quantified by sandwich ELISA.ResultsThe pattern of cytokine concentrations in the supernatants of the stimulated whole-blood cultures was highly individual, but considerably stable over the whole observation period of 4 weeks.ConclusionsBy using TruCulture® it seems feasible to determine subject-specific cytokine reference patterns, for example under healthy conditions, or before starting an experimental treatment, e.g. during a clinical trial, against which changes in the behaviour of the immune system can be detected more accurately in future.

THE MAINTENANCE OF OBLIGATE SEX IN FINITE, STRUCTURED POPULATIONS SUBJECT TO RECURRENT BENEFICIAL AND DELETERIOUS MUTATION

Although there is no known general explanation as to why sexual populations resist asexual invasion, previous work has shown that sexuals can outcompete asexuals in structured populations. However, it is currently unknown whether costly sex can be maintained with the weak structure that is commonly observed in nature. We investigate the conditions under which obligate sexuals resist asexual invasion in structured populations subject to recurrent mutation. We determine the level of population structure needed to disfavor asexuals, as calculated using the average F(st) between all pairs of demes. We show that the critical F(st) needed to maintain sex decreases as the population size increases, and approaches modest levels as observed in many natural populations. Sex is maintained with lower F(st) if there are both advantageous and deleterious mutation, if mutation rates are sufficiently high, and if deleterious mutants have intermediate selective strengths, which maximizes the effect of Muller's ratchet. Additionally, the critical F(st) needed to maintain sex is lower when there are a large number of subpopulations. Lower F(st) values are needed to maintain sex when demes vary substantially in their pairwise distances (e.g., when arrayed along one dimension), although this effect is often modest, especially if some long-distance dispersal is present.

C-Kit mediates chemoresistance and tumor-initiating capacity of ovarian cancer cells through activation of Wnt/β-catenin–ATP-binding cassette G2 signaling

Cisplatin and paclitaxel are standard chemotherapy for metastatic ovarian cancer, but with limited efficacy. Cancer stem/progenitor cells (or tumor-initiating cells, TICs) are hypothesized to be chemoresistant, and the existence of TICs in ovarian cancer has been previously demonstrated. However, the key signals and molecular events regulating the formation and expansion of ovarian tumor-initiating cells (OTICs) remain elusive. Here, we show that c-Kit is not just a marker of OTICs, but also a critical mediator of the phenotype that can be a viable target for the treatment of ovarian cancer. In contrast to non-OICs, c-Kit was overexpressed in OTICs. Moreover, the use of small interfering RNA to inhibit c-Kit expression markedly attenuated the number and size of OTIC subpopulations, inhibited the expression of stem cell markers and decreased the tumorigenic capabilities of OTICs. Imatinib (Gleevec), a clinical drug that blocks c-Kit kinase activity, also demonstrated its inhibition potency on OTICs. In addition, cisplatin/paclitaxel, which killed non-OTICs, with c-Kit knockdown or imatinib revealed that this was critically required for intervening ovarian cancer progression and recurrence in vitro and in xenograft tumors in vivo. Similar results were obtained with OTICs derived from ovarian carcinoma patients. Studies into the mechanisms suggest an important role for the activation of Wnt/β-catenin and ATP-binding cassette G2 downstream of c-Kit. The tumor-promoting microenvironment, such as hypoxia, could promote OTICs via upregulation of c-Kit expression. These results unravel an integral role for c-Kit in ovarian neoplastic processes and shed light on its mechanisms of action.

Role of CD11c-positive dendrite cells in the psoriasis pathogenesis

The immune response, developing with psoriasis relapse, starts from the concentration of activated myeloid dendrite cells, being the main source of the necrosis factor of α -tumor in the area of originating papula. Target: studies of the number of sub-populations of dendrite cells in skin of patients, suffering from psoriasis in different periods of the disease. Маterial and methods. Skin biopsy samples of 43 patients, suffering from psoriasis vulgaris and skin biopsy samples of 16 healthy people have been studied. The skin immunohistochemistry with use of anti-CD11c, CD83 and CD3 аntibodies was performed. Results. The number of CD11c+ and CD83+ dendrite cells in the skin of patients, suffering from psoriasis in the progressing period was 11,2 and 7,8 times more than in the skin of healthy individuals and 3,0 and 2,4 times more than at patients, suffering from psoriasis during remission accordingly. Opinions. In the skin of patients, suffering from psoriasis, during remission at the place of the delivered psoriatic papule the number of CD83+, CD11c+ dendrite cells and CD3+ lymphocytes exceeds its number in the healthy skin, and the ratio of these cells and its localization in the lesion focuses differ from the skin of healthy people.

Population Structure and Genetic Diversity in Popular Rice Varieties of India as Evidenced from SSR Analysis

We report here on the phylogenetic analysis, population substructure, and identification of molecular tags of 25 popular rice varieties and four landraces from different ecological belts of India employing a set of 52 simple sequence repeat (SSR) markers. Genetic analysis using the SSR markers categorized the genotypes into two major clusters, distributed according to their pedigree. Population structure analysis suggested that the optimum number of subpopulations was three (K = 3) in the popular varieties and landraces. At K = 5 the allelic distribution was much more similar to the phylogenetic dendrogram. The molecular diversity and population structure analysis indicated that there is not much variation among the popular rice cultivars of India. The study has identified SSR markers producing unique alleles, which should aid in the precise identification, maintenance, and genetic purity analysis of rice varieties.

Effect of tumor-infiltrating T-lymphocytes on the aggressiveness of epithelial ovarian tumors.

5053 Background: Borderline ovarian tumors (BOT) are a low-grade form of ovarian malignancy with significantly less aggressive behavior than epithelial ovarian cancer (EOC). Since there is neither convincing scientific evidence nor a marker capable of predicting BOT’s behavior, we analyzed the role of immune tolerance in differential disease outcome. EOC and BOT were chosen due to similar disease etiology, despite different disease courses. Increased numbers of T-cell subpopulations infiltrate malignant tumors, so we used epigenetic tests to determine the prevalence of regulatory T-cells (Treg) and overall-T-Lymphocytes (oTL) in EOC and BOT. Methods: The ovarian cancer samples were provided by the European multicentric OVCAD study. The BOT samples were obtained from Tumor Bank Ovarian Cancer at Charité Campus Virchow (Germany). Samples and clinical data were prospectively collected and documented using validated SOPs. DNA was bisulphite-converted and forwarded to methylation-specific RT-PCR for CD3 and FOXP3 loci. Results: We evaluated 90 high-grade EOC, 12 BOT with invasive implants and 25 non-invasive BOT samples. Higher oTL-values correlate with mortality (p=0.008) and recurrence (p<0.001), while higher Treg levels correlate with recurrence (p=0.028). Patients with non-invasive BOT have lower oTL (p=0.019) and Treg (p=0.0005) levels than patients with invasive BOT and EOC, while BOT (both invasive and non-invasive) patients have lower Treg-to-oTL ratios than EOC patients (p=0.0005). Finally, oTL levels associate with overall survival in EOC (p=0.042). Conclusions: We observed a strict correlation between tumor-type, Treg and oTL levels, as well as Treg-to-oTL ratio. This is in agreement with our hypothesis that disease aggressiveness is associated with the amount tumor-infiltrating lymphocytes and may provide the explanation for differing disease courses in EOC and BOT.

Immunologic Response After Laparoscopic Colon Cancer Operation Within an Enhanced Recovery Program

ObjectiveIt has been demonstrated that colon operation combined with fast-track (FT) surgery and laparoscopic technique can shorten the length of hospital stay, accelerate recovery of intestinal function, and reduce the occurrence of post-operative complications. However, there are no reports regarding the combined effects of FT colon operation and laparoscopic technique on humoral inflammatory cellular immunity. MethodsThis was a prospective, controlled study. One hundred sixty-three colon cancer patients underwent the traditional protocol and open operation (traditional open group, n = 42), the traditional protocol and laparoscopic operation (traditional laparoscopic group, n = 40), the FT protocol and open operation (FT open group, n = 41), or the FT protocol and laparoscopic operation (FT laparoscopic group, n = 40). Blood samples were taken prior to operation as well as on days 1, 3, and 5 after operation. The number of lymphocyte subpopulations was determined by flow cytometry, and serum interleukin-6 and C-reactive protein levels were measured. Post-operative hospital stay, post-operative morbidity, readmission rate, and in-hospital mortality were recorded. ResultsCompared with open operation, laparoscopic colon operation effectively inhibited the release of post-operative inflammatory factors and yielded good protection via post-operative cell immunity. FT surgery had a better protective role with respect to the post-operative immune system compared with traditional peri-operative care. Inflammatory reactions, based on interleukin-6 and C-reactive protein levels, were less intense following FT laparoscopic operation compared to FT open operation; however, there were no differences in specific immunity (CD3+ and CD4+ counts, and the CD4+/CD8+ ratio) during these two types of surgical procedures. Post-operative hospital stay in patients randomized to the FT laparoscopic group was significantly shorter than in the other three treatment groups (P < 0.01). Post-operative complications in patients who underwent FT laparoscopic treatment were less than in the other three treatment groups (P < 0.05). There were no significant differences between the four treatment groups regarding readmission rate and in-hospital mortality. ConclusionsThe laparoscopic technique and FT surgery rehabilitation program effectively inhibited release of post-operative inflammatory factors with a reduction in peri-operative trauma and stress, which together played a protective role on the post-operative immune system. Combining two treatment measures during colon operation produced better protective effects via the immune system. The beneficial clinical effects support that the better-preserved post-operative immune system may also contribute to the improvement of post-operative results in FT laparoscopic patients.

Enhanced levels of endogenous common γ chain receptor cytokines influence CD8 T cell subpopulation numbers and phenotype in cancer patients receiving CD4 T cell immunotherapy: Observations In Four Cases Of Patients With Ovarian Cancer (46.31)

Abstract The common γ chain receptor family of cytokines have been associated with T cell homeostasis, development and survival. In this study, we assessed the therapeutic effects of adoptively transferred CD4 T cells and their affects on endogenous expression of such cytokines in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4 effector cell generation, we show that 3 monthly treatment cycles of autologous T cell restimulation and local intraperitoneal reinfusion resulted in diminished serum CA125 tumor marker levels and enhanced patient survival. One patient remains disease free, another patient experienced prolonged survival for nearly 16 months with recurrent disease and two patients expired within 3-5 months following treatment. Prolonged survivors showed lower levels of systemic memory (CD8+CD45RO+)/naïve (CD8+CD45RA+) CD8 T cell ratios when compared to that of short-term survivors. Such subpopulations among prolonged survivors were predominantly FoxP3-NEG and at relatively greater levels when compared to that of corresponding pre-treatment conditions. Lastly, peptide-restimulated PBMCs from these patients showed variable gene expression levels of pre- and post-treatment IL-15, IL-21 and IL-9 that were further associated with prolonged and/or disease-free survival. This suggests that CD4 cell transfer may induce levels of endogenous cytokines associated with CD8 survival and/or homeostasis that may contribute to enhanced CD4 therapeutic potentials

Blood T-cell receptor diversity decreases during the course of HIV infection, but the potential for a diverse repertoire persists

HIV infection results in a decrease in circulating CD4(+) T-cell and naive T-cell numbers. If such losses were associated with an erosion of T-cell receptor (TCR) repertoire diversity in the peripheral T-cell pool, this might exacerbate the state of persistent immunodeficiency. Existing methods for the analysis of the TCR repertoire have demonstrated skewed distributions of TCR genes in HIV-infected subjects but cannot directly measure TCR diversity. Here we used AmpliCot, a quantitative assay based on DNA hybridization kinetics, to measure TCR diversity in a cross-sectional comparison of 19 HIV-infected persons to 18 HIV-uninfected controls. HIV-infected persons had a 10-fold decrease in total TCR repertoire diversity in 1.5 mL of blood compared with uninfected controls, with decreased diversity correlating most closely with a lower CD4(+) T-cell percentage. Nonetheless, the TCR repertoire diversity of sort-purified T-cell subpopulations in HIV-infected and HIV-uninfected subjects was comparable. These observations suggest that the TCR repertoire diversity changes in whole blood during HIV disease progression are primarily the result of changes in the number and proportion of T-cell subpopulations and that most HIV-infected persons may retain a sufficiently diverse TCR repertoire to permit immune reconstitution with antiretroviral therapy alone, without thymopoiesis.

Effects of Korean red ginseng on cyclophosphamide‐induced immunosuppression: comparative effects of 6‐year and 4‐year old Korean red ginseng

In this study, the protective effects of Korean red ginseng (KRG) against cyclophosphamide (CY)‐induced immunosuppression were investigated in animal models. Male BALB/c mice were divided into 6 groups: Normal and CY‐control groups were fed normal diet and the four test groups received modified diets containing 5 or 10% of 4‐year old or 6‐year old KRG powder for 4 weeks. In the subtype and NK cell activity assay, CY was given intraperitoneally in a single dose of 50 mg/kg three days before sacrifice. For the T‐dependent antibody response, CY was given intraperitoneally in a single dose of 50 mg/kg five days before sacrifice and mice were immunized with sheep red blood cells (SRBCs) intraperitoneally in the next day.CY, as reported, showed suppressed effects on body weights, organ weights, splenic lymphocyte subpopulation (total T cells, CD4+ T cells, CD8+ T cells, macrophages and B cells), antibody‐forming cells to SRBC and NK cell activity in these animals. The administration of KRG powder elevated the depressed relative weights of thymus and spleen and increased the number of splenic lymphocyte subpopulation, anti‐SRBC producing cells that were remarkably decreased by the treatment of CY. 6‐year old KRG powder showed more potent effect than 4‐year old KRG in enhancing immune function in the CY‐immunosuppressed mice. These results suggest that 6‐year old KRG is more potent in stimulating immunity in immunosupressed condition than 4‐year old KRG.

Investigating the multimodality of multivariate data with principal curves

We propose a simple method to assess the number of subpopulations in multivariate data by projecting the data on its principal curve and then applying Silverman’s bandwidth test to the resulting univariate sample. Our results indicate that this method works well even in high-dimensional settings with relatively small sample sizes, provided that the number of subpopulations is not large compared to the number of dimensions.

Context underlying decision-making on parenthood and reproduction

Abstract This article provides an overview of a number of research studies conducted within the field of parenthood and reproduction in a variety of Western cultures, including Slovakia and the countries of Eastern Europe. The main aim of this overview is to analyse two key indicators on Second Demographic Transition: delaying marriage and parenthood until later on in life and the growth in cohabitation as an alternative living arrangement and childbearing as part of that. The author points out that the majority of parents opt to have a first child and this brings normative and emotional fulfilment. Most young people do not reject this, but simply postpone it until later on in life. The hypothesis is postulated that the decision on whether to have further children is to a larger extent rationally based and is more dependent on weighing up the incentives available from the state. The article emphasises, on the basis of a comparison of the results of various empirical studies, that the same patterns of reproductive behaviour can produce markedly different consequences and outcomes within various subgroups of the population. It is recommended that current research into parenthood and reproduction should be extended so that studies are conducted into similar groups within subpopulations across different countries—in contrast to the current prevailing research that is focused on single countries or in comparing different countries as a whole. It is also recommended that the scope of the research be extended to compare a number of different subpopulations with similar value and life-style configurations as opposed to the majority of the current narrowly-focused approaches that concentrate on examining the differences in reproductive behaviour patterns of a section of the population selected on the basis of education.

Incidence and risk factors associated with chlamydia in men who have sex with men: a cohort analysis of Victorian Primary Care Network for Sentinel Surveillance data

Chlamydia is the most commonly notified sexually transmitted infection (STI) in Australia. Incidence studies of chlamydia in men who have sex with men (MSM) are rare and offer important public health information. To determine chlamydia incidence in MSM presenting at high caseload clinics and describe predictors of infection. The Victorian Primary Care Network for Sentinel Surveillance of bloodborne viruses and STIs (VPCNSS) links testing, demographic and behavioural data from individual testers at participating clinics. Data from MSM with more than one chlamydia test at the VPCNSS site between April 2006 and June 2010 were included. Chlamydia incidence per 100 person-years (PY) was calculated and Cox regression used to examine predictors of incidence. 1206 positive tests for chlamydia were detected among 6333 MSM across 11,409 PY of follow-up. Overall chlamydia incidence was 10.6/100 PY (95% CI 10.0 to 11.2) and was highest among MSM aged 16-29 years (12.9/100 PY, 95% CI 11.7 to 14.1), presenting with STI symptoms (16.0/100 PY, 95% CI 14.2 to 18.0), HIV positive (18.5/100 PY, 95% CI 16.6 to 20.6) and self-identified sex workers (14.3/100 PY, 95% CI 10.0 to 20.6). Significant predictors of chlamydia infection among MSM were younger age (adjusted hazard ratio (aHR)=1.9, 95% CI 1.5 to 2.3), self-identifying as a sex worker (aHR=1.6, 95% CI 1.0 to 2.6), being HIV positive (aHR=2.6, 95% CI 1.8 to 3.8), presenting with STI symptoms (aHR=1.7, 95% CI 1.4 to 2.1) and reporting >10 sex partners in the past 6 months (aHR=2.5 95% CI 1.4 to 4.6). These results show that MSM represent a key risk population for chlamydia in Australia and identify a number of high-risk MSM subpopulations for whom clinical and public health interventions are warranted.

Differentiation of follicular cells in polytrophic ovaries of Neuroptera (Insecta: Holometabola)

Mechanisms that underlie differentiation and diversification of the ovarian follicular epithelium in insects have been best characterized in a fruit fly, Drosophila melanogaster. Recent comparative analyses have shown that dipterans evolved a common, specific system of early patterning of their follicular epithelium, while some of the follicular cells acquired an ability to undertake active and invasive migrations. To gain insight into the evolution of the differentiation pathways we extended comparative analyses to Neuroptera, one of the most archaic holometabolan insects with polytrophic ovaries. Here, we show that the follicular cell differentiation pathway in neuropteran ovaries significantly differs from that observed in Drosophila and its relatives. In neuropteran ovaries differentiation of the germ line cells precedes the organization of the follicular epithelium. In consequence, at early stages of egg chamber formation germ cell clusters are not enveloped completely by the regular follicular epithelium but associate with two types of somatic cells: interstitial and prefollicular cells. Interstitial cells do not contribute to the formation of the follicular epithelium, while prefollicular cells diversify into a number of follicular cell subgroups. Some follicular cells remain in contact with the nurse cell compartment. The remaining ones associate with the lateral aspects of the oocyte and diversify into the mainbody follicular cells and the anterior and posterior centripetal cells. In the advanced stages of vitellogenesis protrusions of the anterior and posterior centripetal cells penetrate the nurse cell-oocyte interface and dragging behind their neighboring mainbody cells, eventually encapsulate the oocyte pole(s) with a confluent epithelial layer. The follicular cells in neuropteran ovaries are not migratory at all. They may only change their position relative to the germ line cells. Almost complete immobility of follicular cells in neuropteran egg chambers results in a lower number of diversified subpopulations when compared to Drosophila and other true flies.

Improved Eigenanalysis of Discrete Subpopulations and Admixture Using the Minimum Average Partial Test

Principal components analysis of genetic data has benefited from advances in random matrix theory. The Tracy-Widom distribution has been identified as the limiting distribution of the lead eigenvalue, enabling formal hypothesis testing of population structure. Additionally, a phase change exists between small and large eigenvalues, such that population divergence below a threshold of F_ST is impossible to detect and above which it is always detectable. I show that the plug-in estimate of the effective number of markers in the EIGENSOFT software often exceeds the rank of the sample covariance matrix, leading to a systematic overestimation of the number of significant principal components. I describe an alternative plug-in estimate that eliminates the problem. This improvement is not just an asymptotic result but is directly applicable to finite samples. The minimum average partial test, based on minimizing the average squared partial correlation between individuals, can detect population structure at smaller F_ST values than the corrected test. The minimum average partial test is applicable to both unadmixed and admixed samples, with arbitrary numbers of discrete subpopulations or parental populations, respectively. Application of the minimum average partial test to the 11 HapMap Phase III samples, comprising 8 unadmixed samples and 3 admixed samples, revealed 13 significant principal components.

Linkage disequilibrium in a population undergoing periodic fragmentation and admixture

Glacial and interglacial cycles are considered to have caused the fragmentation and admixture of populations in many organisms. A simple model incorporating such periodic changes of the population structure is analysed in order to investigate the behaviour of neutral genetic variation at one and two loci. The equilibrium is reached very quickly in terms of cycles if the length of a cycle is long, as would be expected of the glaciation cycles. Heterozygosity and linkage disequilibrium are shown to depend on the length of time of the fragmented and admixed phases, population sizes, and number (n) of subpopulations in the fragmented phase. If the population size is small in the fragmented phase and its duration is long, the squared correlation coefficient of two loci (a measure of linkage disequilibrium) just after the admixture is approximated by 1/(n-1) for n > 1. After admixture, the correlation decays at a rate of approximately twice the recombination rate. Therefore, if post-glaciation admixture created linkage disequilibrium, we expect to observe linkage disequilibrium even between moderately linked loci, and its decay pattern along the chromosome is very different from that in a random mating population at equilibrium. This is especially true in organisms with long generation times such as trees.

The Interplay of Catalytic Activity and Substrate Promiscuity for an Intrinsically Disordered Staphylococcal Nuclease

In order to investigate the effect intrinsic disorder has on catalytic activity and enzyme-substrate promiscuity, we chose to study one N-, one C- and one N- and C-terminally truncated variant of Staphylococcal nuclease (SNase). SNase is a 149amino acid 3’ endo-exonuclease that is stable, refolds, and retains 75% of native activity in 5M Urea (Jackson and Chalkley, 1975). As SNase is well characterized biophysically and biochemically, comparative analyses of the effects of truncations on stability and catalytic activity, is feasible. By interfering with tertiary structure (N-terminal truncation), secondary structure (C-terminal truncation) or both, the degree of intrinsic disorder was altered, as measured by using circular dichroism, ANS fluorescence and 1D NMR. Following biophysical analyses, the catalytic activity on natural, and non-natural substrates was investigated. Using fluorescence spectroscopy with TAMRA and FAM labeled substrates, we found that the least structured variant; containing both N-terminal and C-terminal truncated regions, was the most active enzyme; exhibiting near native activity for the cleavage of an A16-mer. The order of catalytic efficiency for the substrates was similar between the variants. Nevertheless, introduction of phosphothioate linkages into the A16-mer - hypothesized to be nuclease resistant - was cleaved more efficiently than a C10-mer. No measurable activity was found for the two non-natural substrates chosen (TNA and pRNA). As intrinsic disorder is hypothesized to increase the number of protein sub-populations, with changed dynamics and kinetics as a consequence, bulk measurements are suboptimal to truly measure the actual catalytic efficiency. The results from this study serve as a good primer, enabling the choice of substrates with suitable turnover rates, for future single molecule FRET analyzes into intrinsically disordered SNase.

Estimation of finite mixtures with symmetric components

It may sometimes be clear from background knowledge that a population under investigation proportionally consists of a known number of subpopulations, whose distributions belong to the same, yet unknown, family. While a parametric family is commonly used in practice, one can also consider some nonparametric families to avoid distributional misspecification. In this article, we propose a solution using a mixture-based nonparametric family for the component distribution in a finite mixture model as opposed to some recent research that utilizes a kernel-based approach. In particular, we present a semiparametric maximum likelihood estimation procedure for the model parameters and tackle the bandwidth parameter selection problem via some popular means for model selection. Empirical comparisons through simulation studies and three real data sets suggest that estimators based on our mixture-based approach are more efficient than those based on the kernel-based approach, in terms of both parameter estimation and overall density estimation.

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin Lüscher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin Lüscher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs. Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex. Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.