s2fieldsofstudy: Biology Engineering

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Immunomodulation of TH2 biased immunity with mucosal administration of nanoemulsion adjuvant

Highlights • Nanoemulsion vaccine adjuvant induces robust antigen specific TH1-biased immunity.• Nanoemulsion vaccine adjuvant suppresses established TH2 immunity.• Efficacy of nanoemulsion vaccine in mice with pre-existing immunity to same antigen.• Nanoemulsion vaccines induce IL-10 and regulatory T cells.

Introduction
CD4 + effector T cell responses are classified according to their cytokine and transcription factor profiles, with T H 1 and T H 2 cells being the most widely studied types [1]. Differentiation of T H 1 cells is driven by IL-12 secreted by macrophages and IFN-c from T cells or NK cells, and results in the production of T H 1-type cytokines includ-ing IFN-c, IL-2 and TNF-a. In mice, IFN-c enhances immunoglobulin class switching to increase production of IgG2a and IgG2b subclasses as well as activation of other T H 1 cell-mediated effector responses [2]. Alternatively, T H 2 responses can be initiated by IL-4-dependent differentiation of T H 2 effector CD4 + cells that produce T H 2-type cytokines, including IL-4, IL-5, IL-9 and IL-13, which can culminate in the increased production of IgG1 subclass and IgE antibodies. The T H 1/T H 2 paradigm is useful for classification of immune responses and becomes better defined as mechanisms of action of CD4 + effector T cells are further elucidated.
The type of cell-mediated immunity affects the induction of specific protective immunity to infectious diseases, inflammatory responses, allergy or autoimmunity and even can increase susceptibility to certain infections [3,4]. This is of particular importance in vaccine development because adjuvants are able to skew helper T cell profiles, and choosing the appropriate adjuvant may influence efficacy [5]. The most widely used adjuvant alum induces strong T H 2-associated immune responses which are less effective against pathogens for which T H 1 cell-mediated immunity is required for clearance [6][7][8] Abbreviations: alum, aluminum hydroxide; cLN, cervical lymph node; ELISA, enzyme-linked immunosorbent assay; HBs, hepatitis B surface antigen; i.m., intramuscular; i.n., intranasal; i.p., intraperitoneal; NE, nanoemulsion; ova, ovalbumin; Treg, regulatory T cell.
of vaccines based on new T H 1 polarizing adjuvants including liposomes, CpG-containing oligodinucleotides, monophosphoryl lipid A, and QS-21 are being evaluated both in animal studies and in clinical trials [9][10][11][12]. Many of these adjuvants are under development for production of vaccines that may be used in people who have already been exposed to the same antigen or pathogen, either through prior vaccination or infection. In the case of individuals previously primed to have a T H 2 skewed immune response from an alum-adjuvanted vaccine, it is unclear if boosting with a T H 1-polarizing vaccine adjuvant would redirect the immune response towards a T H 1 response, or if it would simply boost the T H 2 responses for which the immune system had already been primed. Additionally, a considerable interest has been directed towards development of strategies for modulation of existing T H 2 immune responses, especially for the alleviation of T H 2-biased allergic responses [13,14]. Adjuvants capable of redirecting established antigen-specific T H 2 responses to induce T H 1 while suppressing T H 2 immunity have the potential for impacting a variety of diseases driven by aberrant T H 2 immune responses.
Our group has developed a nanoscale oil-in-water emulsion (nanoemulsion, NE) vaccine adjuvant platform that when delivered intranasally (i.n.) induces robust systemic and mucosal responses without local inflammatory effects [15][16][17][18][19][20]. In animal studies, i.n. immunizations with NE mixed with a variety of viral and bacteria-derived antigens, including influenza, hepatitis B, respiratory syncytial virus, vaccinia and anthrax, yields high protective antibody titers. In contrast to adjuvants like alum that induce T H 2-biased immune responses [6,21,22], nasally administered NE vaccines result in T H 1 and T H 17 polarized immune responses [18,23,24]. Regardless of the model tested, T H 2 cytokine responses in animals immunized with NE are always low, and no significant production of IgE has been observed. This is true even in BALB/c mice that are inherently biased towards a T H 2-type response [25]. Because this NE adjuvant is a robust T H 1-polarizing adjuvant, we hypothesized that it would be a good candidate for redirecting established T H 2 immune responses to a more balanced T H 1/T H 2/ T H 17. In proof-of-concept studies presented here, we have investigated the effect of nasal administration of a NE vaccine in mice previously vaccinated with an alum-adjuvanted vaccine.

Antigen and adjuvants
The recombinant hepatitis B surface antigen (HBs) was supplied by Human Biologicals Institute (Indian Immunologics, Ltd, Hyderabad, India). The endotoxin level was determined to be <7.5 EU/20 lg of HBs, which is significantly below the internationally accepted standard of 630 EU/20 lg of protein. Ovalbumin (ova) was purchased from Sigma-Aldrich. Ova peptides, class I-restricted ova 257-264 (SIINFEKL, ova I) and class II-restricted ova 323-339 (ova II) were purchased from Invitrogen. Nanoemulsion adjuvant (NE) was supplied by NanoBio Corporation, Ann Arbor, MI. NE was produced by a high speed emulsification of ultra pure soybean oil with cetyl pyridinium chloride, Tween 80 and ethanol in water, with resultant NE droplets with average 350-400 nm diameter [18]. Aluminum hydroxide (alum) was purchased from Sigma-Aldrich, Inc. All reagents were tested for the presence of endotoxin using RAW-Blue cell-based assay in vitro (InvivoGen, San Diego, CA).

Mice and immunizations
Pathogen-free CD-1 mice (females 6-8 weeks old) were purchased from the Charles River Laboratories. All animal procedures were performed according to the University Committee on the Use and Care of Animals at the University of Michigan. Immunization schedule is shown in Fig. 1. For all immunizations, mice were anesthetized under isoflurane anesthesia using the IMPAC6 precision vaporizer. Intranasal (i.n.) immunizations were done using a pipette tip by administration of 5 ll/nare of formulation containing 20 lg of antigen mixed with 20% NE. Antigen mixed with PBS alone served as a control. Intramuscular immunizations (i.m.) were performed by injection of 50 ll containing 20 lg of antigen adsorbed on 0.5 mg/ml alum into the epaxial muscle as described previously [18]. Sera were obtained by saphenous vein bleeding, and splenocytes were harvested at the end of the experiment. In IL-10 depletion experiments, mice were injected i.p. with 1 mg anti-IL-10 (purified from rabbit serum [26]) or control rabbit IgG 12 h before and 2 days after NE immunization.

Measurement of serum IgG subclasses
Serum antibody and IgG subclasses titers were determined by ELISA, with plates coated with 5 lg/ml of HBs as described previously [18].

Analysis of cytokine expression
Single cell suspensions of freshly isolated mouse splenocytes were cultured at 4 Â 10 6 cells/ml with or without antigen (10 lg/ml). After 48 h, supernatants were collected and analyzed for the presence of cytokines using Milliplex Mouse Cytokine/ Chemokine Immunoassay Kit (Millipore, Billerica, MA).

Measurement of the induction of regulatory T cells (Tregs) after NE immunization
Mice were immunized i.n. with ova and NE (ova-NE) or non-adjuvated ova (ova-PBS) at weeks 0 and 4. Splenocytes were harvested at 1 and 7 weeks after the first immunization. Red blood cell depleted single cell suspensions were stained by flow cytometry to quantify regulatory T cells. Fc receptors were blocked with purified anti-CD16/32 (clone 93, BioLegend) and surface markers were stained using antibodies against CD3 (145-2C11), CD4 (RM4-5) and CD25 (7D4) (all from eBioscience or BD Biosciences), permeabilized, fixed and labeled for intracellular Foxp3 (FJK-16s). Samples were acquired on an Accuri C6 flow cytometer (BD Biosciences). Data were analyzed using FlowJo (Treestar).

Statistics
Results are presented as the geometric mean ± 95% confidence interval. Statistical comparisons were assessed by the Mann-Whitney test using GraphPad Prism version 6 (GraphPad Software). The p value < 0.05 was considered as significant. In every reported result the data shown are representative of at least 2 experiments.

Mucosal immunization with NE adjuvant modifies T H 2 polarized immune response
To elicit the T H 2 response, mice were immunized with two i.m.
injections of 20 lg HBs adsorbed on alum [21]. Analysis of serum IgG subclass and cytokine expression confirmed that HBs-alum immunization yielded predominantly IgG1 antibody subclass ( Fig. 2A) and induction of T H 2-type cytokines IL-4 and IL-5 (Fig. 2B). There was no change in antibody titers in mice receiving only the HBs-alum vaccine from weeks 6-12 (data not shown). To investigate whether NE adjuvant can modify this T H 2 bias, the mice were subsequently immunized with a single intranasal administration of HBs-NE at 2 or 6 weeks after the second HBs-alum sensitization (Fig. 1). Serum IgG analysis showed significant increases in IgG2a and IgG2b subclasses following HBs-NE immunization, with antibody titers comparable to the HBs-NE immunization in mice that did not receive the HBs-alum vaccine ( Fig. 2A). Antigen-specific cytokine expression in splenic lymphocytes after the 6 week NE immunization showed significant induction of T H 1-type IFN-c and TNF-a and the T H 17 cytokine IL-17 ( Fig. 2B) and decreased IL-4 and IL-5 production in mice immunized with HBs-NE six weeks after HBs-alum sensitization. This effect was not significant in mice immunized with HBs-NE at an earlier time point (2 weeks). Nasal immunization with HBs-NE alone was used as a control to assess modulation of established T H 2 immunity with NE adjuvant, and antibody and cytokine patterns were similar after HBs-NE immunization regardless of whether the mice had been previously T H 2 sensitized or not ( Fig. 2A and B). There was a slight decrease in IFN-c and IL-17 in mice that received both vaccines compared with mice immunized with NE only, however these differences were not statistically significant (p = 0.70 and 0.41, respectively).
To investigate a potential role of regulatory T cells (Tregs) in the mechanism of NE adjuvant, mice were immunized i.n. with ova-NE or with non-adjuvanted ova in PBS (ova-PBS) as a control. Treg frequency (CD4 + Foxp3 + ) was measured after 6 days both in the nasal draining lymph nodes (cervical lymph nodes, cLN) and in the periphery in splenic lymphocytes. Analysis of CD4 + Foxp3 + T cells showed that mucosal administration of ova-NE induced significantly more Treg expansion in both cLN and spleen compared to ova-PBS and PBS administration (p < 0.03) (Fig. 3A). Interestingly, by 6 weeks after immunization the frequency of Tregs was elevated in both ova-NE and ova-PBS groups (Fig. 3B). Consistent with results documented previously, the i.n. immunization with ova-NE induced a potent IgG response, while no significant titers were detected in ova-PBS immunized mice (Fig. 3C).
Further analysis revealed that production of IL-10, a suppressive cytokine associated with Treg function [27,28], was increased in cells from mice immunized with i.n. ova-NE (Fig. 4A). Furthermore, IL-10 production was only significantly induced with stimulation with ova protein or a MHC II-restricted ova peptide, not a MHC I ova peptide, suggesting that the IL-10 is produced by CD4 + T cells. Correlates of Treg frequency vs. IL-10 expression show no IL-10 production in mice immunized with ova-PBS despite the increase in frequency of Treg frequency. In contrast, in mice treated with ova-NE there was a significant increase in IL-10 levels that closely correlated with increased Treg frequency (Fig. 4B).
In order to determine the effects of IL-10 on NE-mediated suppression of the alum-induced T H 2 immune response, mice were immunized i.m. with HBs-alum and IL-10 was depleted at the time of HBs-NE immunization. There was no balancing of IgG subclasses when IL-10 was depleted during NE immunization (Fig 5A), and the subclass pattern was similar to that observed from mice only receiving the HBs-alum vaccine. The suppression of T H 2 cytokines (IL-4, IL-5, IL-13) did not occur upon HBs-NE immunization with simultaneous IL-10 depletion. The induction of the T H 1 cytokine, IFN-c, was not inhibited by IL-10 depletion. IL-10 depletion did not significantly change the percentage of Tregs induced by NE immunization.

Discussion
The development of new materials and adjuvants that can modulate the immune system is an emerging field in immunology, with interests in multiple settings, including vaccine development and allergy [13,[29][30][31]. In this proof of concept study we present a new adjuvant-based approach to immunomodulation in mice. We have demonstrated that immunization with novel oil-in-water nanoemulsion adjuvant not only produced robust cellular and humoral immunity but also redirected existing T H 2-biased responses towards a more balanced T H 1/T H 2 phenotype in a model of established antigen-specific T H 2 immunity.
In contrast to the commonly used aluminum adjuvant(s), NE is not associated with the T H 2 phenotype. Consistent with our previous results [16][17][18], immunization with NE adjuvant produced T H 1 biased immunity with IFN-c and TNF-a production (Fig. 2B). The significant increase of IgG2a and IgG2b antibodies and T H 1 type cytokines and simultaneous reduction of IgG1 antibodies and T H 2 cytokines demonstrates that NE adjuvant is capable of shifting an established T H 2 response towards a more balanced cell-mediated immunity both through the induction of T H 1 and suppression of T H 2. In mice, IgG1 is regulated via a T H 2/IL-4 pathway, and in numerous studies IgG1 has been used as a robust indicator for the assessment of a T H 2 response [32,33]. Mucosal HBs-NE immunization of HBs-alum sensitized mice diminished IgG1/IgG2a and IgG1/IgG2b ratios from 10.46 and 8.67, to 1.2 and 2.1, respectively, clearly demonstrating modulation of the HBs-specific immune response.
Analysis of cytokine expression provided direct assessment of T cell activation. Elevated IFN-c and diminished IL-4 levels after antigen stimulation of splenic lymphocytes indicated that HBs-NE immunization resulted in T H 2 cell suppression and a shift to T H 1 response (Figs. 2B and 3B). This effect was not detected in splenocytes from mice immunized with NE at an earlier time point (2 weeks), despite increase in IgG2a and IgG2b antibodies in comparison to HBs-alum controls (Fig. 2). This result indicates that while NE has the potential to modify established T H 2 immunity, effective modification of an ongoing immune response may depend on the schedule and number of immunizations.
Intranasal immunization with NE adjuvant induces a T H 17 immune response [23]. The antigen-specific IL-17 expression was also detected in splenocytes of mice with T H 1 redirected immune response (Fig. 2B). Despite association with various autoimmune disorders, T H 17 also contributes to host defense as a T cell subset involved in protection against extracellular pathogens [34] and has been shown to play a critical role in the efficacy of several vaccines [35][36][37][38][39]. Although excessive prolonged IL-17 production may contribute to pathophysiology of respiratory infections or asthma and allergy, the degree of T H 17 induction with NE immunization is much lower than levels typically observed in diseases in which IL-17 contributes to pathology [40,41]. The effect of NE-induced IL-17 production on T H 2/T H 1 immunomodulation remains to be investigated; however, T H 17 cell-mediated immunity may suppress IgE responses, as has been recently indicated for T H 17 immunity associated with human autoimmune disease [42].
The exact mechanism of action of NE adjuvant is not yet fully elucidated. NE is formulated using ultrapure and endotoxin-free components and does not contain any commonly recognized TLR agonists or ligands [18]. However, our recent results demonstrate involvement of the TLR pathway in immunogenicity of NE adjuvant both in vivo and in vitro [24]. NE facilitates antigen uptake and trafficking into lymphoid tissue while not causing either nasal irritation or disruption of mucosal epithelial architecture [43,44]. NE-mediated enhancement of antigen internalization and processing by the antigen presenting cells could be important for the optimal antigen presentation to T cells and development of T H 1 biased immunity [45][46][47]. We have shown that intranasal treatment with NE adjuvant does not produce significant amounts of IFN-c, TNF-a, IL-12, IL-4, IL-5, IL-9, IL-13 or inflammatory cytokines such as IL-1b in the nasal mucosa [43]. Based on the absence of inflammatory mediators, rhinitis or cellular infiltrates at the high 20% concentration, NE appears to be non-inflammatory and is generally biocompatible with mucosal and pulmonary tissue in mice, rats, guinea pigs, dogs and humans (not shown and [18,44,48]).
While data from mouse models clearly show that alum drives T H 2 immunity, the evidence for T H 2 skewing by alum based vaccines in humans is not entirely clear. A few clinical studies have shown that alum induces a mixed T H 2 and T H 1 response, but the overall effect across various antigens in humans as compared to mice is poorly defined [49][50][51][52]. Additionally, studies assessing immune polarization induced by alum mainly have been performed in adults. Given that neonatal immune systems are inherently biased towards T H 2 [53,54], the immunization of newborns with an alum-based Hepatitis B vaccine raises concerns about the role vaccines might play in the growing issue of allergic disease in young people [55,56]. Moving forward, it may be advantageous to consider vaccine adjuvants that induce required protective immunity without activating T H 2 polarized responses. While the ability of NE adjuvant to shift towards T H 1 in humans is not explored in this study, in a Phase I clinical trial a flu vaccine containing this NE adjuvant formulation induced T H 1 antigenspecific IgG, neutralizing antibody, as well as mucosal IgA, demonstrating the immunogenicity of this adjuvant in humans [44].
Here, we demonstrate that NE immunization resulted in the induction of Tregs in both the draining lymph nodes and the periphery. The correlating increase in IL-10, suggests that these Tregs may have suppressive function, and likely play a role in the immune responses induced by NE [57]. Tregs are considered essential for the maintenance of immunological homeostasis and for the control of exacerbated immune responses. Numerous studies have demonstrated a role for Tregs in restraining exacerbated immune responses during natural infection, suggesting that Treg depletion and/or inactivation could improve efficacy of vaccines [58,59]. Much less is known regarding the role of Tregs in the induction and maintenance of protective immune response with various adjuvant-based vaccines; however the data presented here suggest that for NE the induction of Tregs does not inhibit overall vaccine efficacy but may be responsible for the suppression of the T H 2 response. It has previously been reported that antigen-specific T H 1 and regulatory T cells can mediate modification of IgG subclass pattern [60], consistent with the data presented here. Since Tregs induced with antigen alone are often considered as immune suppressors in the process of immune tolerance, our results may suggest a functional difference between the Treg populations in mice immunized with antigen alone compared with antigen and NE. Similarly, these results may suggest a functional difference between Treg populations generated in various modes of i.n. immunization. Further characterization of Treg function and direct functional assessment of their suppressive potential will help to clarify their role in NE induced immune response.
IL-10 production is one mechanism by which immune responses can be suppressed. Not only does NE induce IL-10, but depletion of IL-10 during NE immunization alters the ability of NE to suppress T H 2 immunity (Fig. 5). Interestingly, IL-10 depletion did not alter T H 1 induction by NE, so IL-10 does not appear to be involved in the induction of immune responses by NE. IL-10 was depleted during immunization but not at the time of sacrifice when the recall response to antigen was determined, suggesting that IL-10 is critical for priming of cellular immune responses that result in a shift from T H 2 to T H 1 in this model.

Conclusions
Our initial results suggest the usefulness of NE-based delivery systems in the development of therapeutic vaccines to modify T H 2 immune responses, as well as the ability of NE-based vaccines to retain their immune phenotype even in individuals that received previous vaccinations with the same antigen and other adjuvants. This novel approach to immunomodulation using i.n. delivery of NE adjuvant to produce mucosal immunity and a systemic T H 1-biased immune response could be useful for the development of vaccines to induce antigen-specific T H 1 immune responses even in individuals with pre-existing T H 2 biased immunity. This suggests that NE adjuvant may be especially useful in situations where pathologies are due to aberrant T H 2 immune response, such as allergy.

Grant support
This project has been funded by the Bill and Melinda Gates Foundation, under award 37868 and the National Institute for Allergy and Infectious Disease, National Institutes of Health under Contract No. HHSN272200900031C.


extfieldsofstudy: Biology Medicine

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Characterizing Variation of Branch Angle and Genome-Wide Association Mapping in Rapeseed (Brassica napus L.)

Changes in the rapeseed branch angle alter plant architecture, allowing more efficient light capture as planting density increases. In this study, a natural population of rapeseed was grown in three environments and evaluated for branch angle trait to characterize their phenotypic patterns and genotype with a 60K Brassica Infinium SNP array. Significant phenotypic variation was observed from 20 to 70°. As a result, 25 significant quantitative trait loci (QTL) associated with branch angle were identified on chromosomes A2, A3, A7, C3, C5, and C7 by the MLM model in TASSEL 4.0. Orthologs of the functional candidate genes involved in branch angle were identified. Among the key QTL, the peak SNPs were close to the key orthologous genes BnaA.Lazy1 and BnaC.Lazy1 on A3 and C3 homologous genome blocks. With the exception of Lazy (LA) orthologous genes, SQUMOSA PROMOTER BINDING PROTEIN LIKE 14 (SPL14) and an auxin-responsive GRETCHEN HAGEN 3 (GH3) genes from Arabidopsis thaliana were identified close to two clusters of SNPs on the A7 and C7 chromosomes. These findings on multiple novel loci and candidate genes of branch angle will be useful for further understanding and genetic improvement of plant architecture in rapeseed.

INTRODUCTION
Rapeseed (Brassica napus L. 2n = 4 × = 38, AACC genomes) is a widely cultivated oil crop throughout the world. Yield improvement and mechanized harvesting are extremely urgent recently for the demands of rapeseed producers in addition to the edible oil and biofuel industries (Diepenbrock, 2000). The ideotype of a plant is defined as the spatial distribution of various architectures which is an important agronomic character that affects photosynthesis and seed yields (Donald, 1968;Mansfield and Mumm, 2014). The ideotype can influence photosynthesis, plant growth, and seed yield due to the least competition among the individuals in a population (Wang and Li, 2005). The ideotype is determined by a combination of architecture factors including branch angle (BA), plant height (PH), first branch height (FBH), inflorescence length (IL), and branch number (BN; Mei et al., 2009;Shi et al., 2009;Xu et al., 2014). In particular, branch angle, or the angle between branch and erect primary stem, has long attracted the attention of breeders because of the significant contribution of this trait to plant architecture.
High yields can be achieved through high plant density with a small branch angle, which determines the plant's ability to grow and capture light efficiently. Wang and Li (2008) reported in rice more upright and dense leaves not only improve light capture but also improve the accumulation of leaf nitrogen for grain filling. The branch (or leave) angle has also been studied in maize, cotton, and other important crops to achieve an ideal plant architecture to improve yields Jin et al., 2008;Song and Zhang, 2009;Ku et al., 2011;Tian et al., 2011;Bai et al., 2012). Several quantitative trait loci (QTL) related to leaf angle have been identified genetically in maize.
In recent years, genome-wide association (GWA) mapping has become a powerful tool for identifying important genes associated with complex traits, which has been used with success in model and non-model plants (Breseghello and Sorrells, 2006;Atwell et al., 2010;Huang et al., 2010;Zhao et al., 2011). Additionally, with the dramatically decreasing cost of genome sequencing and rapid developments in genome analysis, Brassica A genome sequence from Brassica rapa and Brassica C genome sequence from Brassica oleracea have been published (Wang et al., 2011;Liu et al., 2014). It is important to complete the rapeseed (B. napus, AC genome) genome sequencing (Chalhoub et al., 2014). Single nucleotide polymorphisms (SNPs) are abundant and evenly distributed throughout the genomes, which is more conducive to GWA. In 2012, a B. napus 60K SNP Infinium genotyping array was produced and applied by the international Brassica SNP consortium in cooperation with Illumina Inc. San Diego, CA, USA (Snowdon and Iniguez Luy, 2012;Edwards et al., 2013). Studies about GWAS in rapeseed have gained attention in recent years and various traits including flowering time, seed weight and seed quality have been dissected (Harper et al., 2012;Cai et al., 2014;Li et al., 2014;Lu et al., 2014;Raman et al., 2014;Wang et al., 2014). However, no report was found on association mapping for rapeseed branch angle to our knowledge.
Numerous genes are known to influence the branch (tiller) angle in model and other crops. For instance, OsTAC1 was reported to play a critical role in controlling rice architecture (Yu et al., 2007). Li et al. (2007) reported that LAZY1 regulates shoot gravitropism through which the rice tiller angle is controlled. Enlarged branch angles with agravitropic shoots were similarly also found in Atlazy1 mutant of Arabidopsis (Yoshihara et al., 2013). Dong et al. (2013) identified maize ZmLA1 gene as a functional ortholog of LAZY1 in rice and Arabidopsis. The regulation of branch angle is a combination of environmental factors and hormone homeostasis (Lomax, 1997). Auxin may be the primary hormone involved in shoot gravitropism (Robert and Friml, 2009), which is a key process in determining branch (leave) angle. The member of GH3 family plays crucial roles in auxin homeostasis in relation to leaf inclination control (Zhao et al., 2012). Recent evidence showed that new strigolactone plant hormones regulated rice tiller angle by attenuating shoot gravitropism through the inhibition of auxin biosynthesis (Sang et al., 2014). Although interaction of auxin and strigolactone plays an important role in shoot gravitropism, the key genes by which the two hormones regulate shoot gravitropism is not yet identified. However, knowledge of the genes that control genetic variation for branch angle of rapeseed is limited.
In this study, a panel of 143 elite rapeseed accessions was analyzed by the 60 K Brassica Infinium SNP array. The branch angle was measured in 2013-2014 at three environments. The SNPs in the array were in silico mapped to A and C genomes of Darmor-bzh B. napus genome "pseudomolecules" to obtain their hypothetical position. The aims were (1) to gain the population structure and genetic diversity in elite germplasms; (2) to detect QTL controlling branch angle and mine for elite alleles; and (3) to predict the candidate genes.

Plant Material and Field Experiments
A total of 143 rapeseed accessions were used for an association analysis in this study. According to the information from field observations, the accessions were classified to three different germplasm types, i.e., spring oilseed rape (OSR) (13), semiwinter OSR (124), and winter OSR (6). Based on their origins, 112 accessions originated from China, 24 from Oceania, 5 from Europe, 1 from North America, and 1 from India (Supplementary Table 1). The seeds from all the accessions were collected, stored and supplied by Oil Crops Research Institute of Chinese Academy of Agricultural Sciences (OCRI-CAAS). In recent decades, these accessions have been widely used as parents in breeding programs.
The experiments were conducted at Yangluo Agronomic Experimental Station of OCRI-CAAS (28 • 42 ′ N, 112 • 33 ′ E), Wuhan, China, during the 2013 and 2014 winter growing season and at Lu'an Experimental Station (31 • 73 ′ N, 116 • 52 ′ E) in Anhui, China, during the 2013 winter growing season. In each environment, the experiment was conducted in randomized complete blocks with two replicates. Each plot contained three rows with 54 individuals, setting as 33 cm between rows and 11 cm between plants within each row, with a planting density of 270,000 plants/ha. All experiments were performed under local field management and cultivation conditions.

Trait Measurements and Statistical Analysis
In each plot, five typical plants were harvested for branch angle measurement at the mature stage. The branch angle (BA) was measured as the angle between the main stem and the branch. BA was scanned with a digital camera (SONY DSLR-A350; SONY, Japan). The angle value from the images was obtained using AutoCAD software (Autodesk Inc., San Rafael, CA). The average BA value of five individual plants for each plot was calculated as the final phenotypic value. In addition, the best linear unbiased estimators (BLUPs) across all three environments were predicted by assuming fixed genotypic effects to minimize the effects of environmental variation. Finally, each environment and BLUP value was used as a phenotype for the association analysis.
Statistical analysis of the data was performed by using PROC MEANS in SAS software, Version 9.3 (2000, SAS Institute Inc., Cary, NC, US). Analysis of variance (ANOVA) was conducted by using PROC GLM to determine the effects of block, environment, genotype and genotype-environment interactions. Correlation coefficients were obtained by using PROC CORR. The Broad-sense heritability (H 2 ) was calculated as H 2 = σg 2 /(σg 2 + σgl 2 /n +σe 2 /nr), in which σg 2 , σgl 2 , σe 2 , r, and n represent the estimated variances for the genetic effects, genotype-environment interactions, random errors, number of replications and number of environments, respectively. The estimated variances for σg 2 , σgl 2 , and σe 2 were obtained by ANOVA.

SNP Genotyping, Quality Control and In silico Mapping of SNPs
The genotype of 143 accessions was detected with a Brassica 60K Illumina Infinium SNP array according to the work flow by Emei Tongde Co. All of the SNP data were clustered and called up automatically by using Illumina BeadStudio genotyping software. The SNP quality was checked and comparable with previous studies Wang et al., 2014). The low quality SNP loci (call rate < 80% and/or minor allele frequency < 0.05) in all accessions were deleted from the results. Out of 52,157 SNPs in the array, 2836 that had a zero call frequency of AA or BB were excluded according to the quality control. Using a cut-off for missing data >0.2 and MAF < 0.05, 1860 and 1909 SNPs were filtered, respectively, reducing the number of SNPs to 38,063.
SNP mapping was performed as previously reported (Altschul et al., 1990). In brief, BLAST search against the "pseudomolecules" representative of the B. napus genome (version 4). Only the top BLAST hits with an E-value cut-off of 1E-15 against the pseudomolecules were retained, while BLAST matches to multiple loci with the same E-value were deleted. A final set of 34,469 high-performing SNPs was used for all the analyses.

Population Genetic Analysis
Nei's genetic distance matrix for all SNPs is the distance and it was calculated to build unrooted neighbor-joining trees by using PowerMarker (Liu and Muse, 2005). The result was visualized by using FigTree software based on 34,469 SNPs (http://tree.bio. ed.ac.uk/software/figtree/). Kinship (K) matrix used to compare all the pairs of the 143 accessions was calculated with 2434 informative SNPs with a MAF > 0.2 by using the SPAGeDi software package (Hardy and Vekemans, 2002). All negative kinship values that were found between two individuals, which indicates that there was less of a relation than expected between two random individuals, were transformed to zero (Yu et al., 2005).
A total of 2434 SNPs [minor allele frequency (MAF) ≥ 0.2] that were evenly distributed across the whole genome were selected to create the population structure inferred by using the STRUCTURE v2.3.4 software package (Pritchard et al., 2000). Iterations were performed 100,000 times by using a burn-in length of 100,000 MCMC (Markov chain Monte Carlo) with the admixture and related frequency model. Five independent runs were performed with K-values (the putative number of populations) ranging from 1 to 10. The optimal K-value was determined by taking the log probability of the data [LnP (D)] and an ad hoc statistical k based on the rate of change for LnP(D) between successive k-values as described by Evanno et al. (2005). The cluster membership coefficient matrices of replicate runs from STRUCTURE were integrated to obtain a Q matrix by using CLUMPP software (Jakobsson and Rosenberg, 2007) and graphically displayed by using the DISTRUCT software package (Rosenberg, 2004). Accessions with a probability of membership >0.7 were assigned to corresponding clusters, and those <0.7 were assigned to a mixed group. The population structure matrix (Q) was generated for further analyses. Linkage disequilibrium (LD) parameter (r 2 ) for estimating the degree of LD between pair-wise SNPs (MAF ≥ 0.05) was calculated using the software TASSEL 4.0 (Bradbury et al., 2007).

Association Analysis
Two different models were used to test associations. The first model was a simple and general linear model (GLM) without controlling for Q and K, containing only the SNP that was tested as a fixed effect. The second model was a mixed linear model (MLM) where, in addition to testing the SNP, the population structure (Q) and relative kinship matrix were included as fixed and random effects, respectively. Analyses were performed by using TASSEL 4.0 software, for which the optimum compression and population parameters previously determined (P3D) variance component estimation were implemented to decrease the computing time for the large data set (Zhang et al., 2010).
The significance of associations between SNPs and trait was based on a threshold of p < 2.90 × 10 −5 [i.e., -log 10 (p) = 4.5]. The threshold is 2.90 × 10 −5 at a significant level of 1% after Bonferroni multiple test correction (1/34,496). Furthermore, we applied the false discovery rate (FDR) technique. We calculated an FDR q-value for each association test by using the software QVALUE (Dabney and Storey, 2004). The FDR q-value of the significant SNP with the lowest test statistic (P < 0.05) provided an estimate of the proportion of false positives among the significant associations. The significant value and the marker effect for each SNP were exported, and a Manhattan plot was generated in the R package qqman (Turner, 2014, http://cran.rproject.org/web/packages/qqman/).
Stepwise regression was performed to detect the effect of multiple alleles with different functional polymorphisms on branch angle and to estimate the total variance explained (R 2 ) by using the lm function in R (Ihaka and Gentleman, 1996).

Phenotypic Variation of Branch Angle
Significant variation was observed among the 143 rapeseed accessions in the three environments studied, with branch angles ranging from 20 to 70 • (Figure 1, Table 1). In three environments Yangluo-2013, Yangluo-2014, and Lu'an-2013, the natural population exhibited average branch angle values (±SD) of 39.93 ± 5.98, 37.52 ± 7.26, and 42.30 ± 6.65, respectively. The frequency distributions of branch angle in the natural population are summarized in Figure 2. Branch angle ranged from 22.32 to FIGURE 1 | Branch angle was measured using AutoCAD software. The two-way ANOVA showed that differences among the lines in the branch angle were highly significant. This finding confirmed that a large number of genetic variation existed in the population. The effects from years and location were significant ( Table 2). The broad-sense heritability (H 2 ) for branch angle was calculated as 93.90%, demonstrating that branch angle in these rapeseed lines was conditioned primarily by genetic factors. The correlation coefficients for branch angle between environments were all relatively high (r ≥ 0.557, P < 0.01).This observation indicated that branch angle in the 143 lines was relatively consistent across environments.

SNP Genetic Diversity and Linkage Disequilibrium
Supplementary Table 2 gives the information on all of the SNPs. Estimates of an average nucleotide diversity (polymorphism information content or PIC) of 0.366 showed that the overall genetic variation in the accessions studied here represents ∼62.9% of the rapeseed diversity. There had the most markers (2745) with a marker density of one per 26.4 kb in C3 linkage group and the fewest markers (932) with a marker density of one per 47.6 kb in C5.
To evaluate the extent of LD, r 2 was used to calculate LD. The genome-wide LD decay of A and C genome for rapeseed germplasms are shown in Figure 3. Taken together, the LD of A genome decayed significantly faster than that of the C genome. We estimated the LD decay, r 2 decayed to 0.2 when the average distance for A genome was 206 kb and r 2 decayed to 0.2 when the average distance for C genome was 949 kb.

Population Structure and Relative Kinship
Population structure of the association panel was calculated using 2434 SNPs, and a clustering inference performed with possible clusters (k) from 1 to 10 showed that the most significant change in likelihood occurred when K increased from 2 to 3, and the highest k-value was observed at k = 2 (Figure 4). Based on the k method (Figure 4B), the 143 accessions could be divided into two sub-populations ( Figure 4C). By using a probability-of-membership threshold of 70%, 99 and 17 lines were assigned to the two groups, respectively. The remaining 27 lines were classified into a mixed group (Supplementary Table 1). In comparison with a previous study, this population structure classification yields the same results (Harper et al., 2012;Lu et al., 2014;Wang et al., 2014). In addition, the NJ phylogenetic tree based on Nei's genetic distances displayed two clear clades (Supplementary Figure 1), corresponding to the two groups estimated by STRUCTURE. The lines belong to mixed groups were distributed across the whole tree. Tree-based analyses yielded results very similar to those of the STRUCTURE analysis.
The 2434 informative SNPs with a MAF > 0.2 and little or no missing data were used to estimate the relative kinship in the set of 143 lines. As shown in Figure 5, the average relative kinship between any two lines was 0.0332, or ∼57% of the pairwise kinship estimates were close to 0, and 21% of the kinship estimates ranged from 0 to 0.05. The remaining estimates ranged  from 0.05 to 1, with a continuously decreasing number of pairs falling in higher estimated categories. These results indicated that most lines in the panel have no or very weak kinship, which might be attributed to the broad ranging collection of genotypes and the exclusion of similar genotypes before analysis.

Association Mapping and Candidate Gene Prediction
A total of 25 and 60 associations (P < 2.90E −5 ) were detected for branch angle by using BLUP across three environments and an individual environment (Figure 6, Table 3; Supplementary  Figure 2). To select the major QTL among all the significant SNPs, these SNPs were clumped by using an LD block as a criterion (Gabriel et al., 2002), and the peak SNP within each LD block was retained. After the clumping of SNPs, six QTL for branch angle were distinguished with the BLUP values of a branch angle across three environments and the peak SNPs are listed in Table 3. The six peak regions were 6.1, 3.5, 23.2, 3.0, 39.5, and 48.7 Mb of A2, A3, A7, C3, C5, and C7, respectively, in the "pseudomolecules" of B. napus, and the cumulative phenotypic variance explained by all significant SNPs was 92.42%, which contributed to 16.60-18.95% of the phenotypic variance based on the R 2 -values. In Yangluo-2013, nine peak SNPs were detected in Q+K models with an FDR of 0.021, and the eight peak regions were 10. 1, 22.2, 18.0, 0.1, 42.8, 48.7, and 2.9 Mb for A2, A5, A8, A10, C2, C6, C7, and C8, respectively. In Yangluo-2014, two peak SNPs were detected with FDRs of 0.013 and 0.027, and the two peak regions were 48.7 and 23.2 Mb of A7 and C7, respectively. In Lu'an-2013, only one significantly peak SNP in A3 was detected in 31.3 Mb region with an FDR of 0.263. Notably, the Lazy orthologs were searched in the "pseudomolecules" of B. napus and two orthologs were found at 2.6 Mb in A3 and 3.2 Mb in C3, which were 488 kb away from the peak SNP Bn-A03-p3571859 and 243 kb away from the peak SNP Bn-A03-p3571859. In addition, the Squamosa Promoter Binding Protein-like 14 (SPL14) orthologs were searched in the "pseudomolecules" of B. napus and one ortholog was found at 24.3 Mb of A7, which was 521 kb away from the peak SNP Bn-A07-p19977445. Aside from the Lazy and SPL14 genes, an auxin-responsive GRETCHEN HAGEN 3 (GH3) ortholog "pseudomolecules" of B. napus in 48.7 Mb of C7, which was very close (only 4 kb) to the peak SNP Bn-scaff-16110-1-p1940585.

DISCUSSION
In this study, a panel comprising 143 B. napus germplasm lines was adopted for association mapping study. Significant natural phenotypic variation in branch angle was observed in rapeseed germplasms. In general, to unravel the genetic basis of trait variation, a number of traditional genome-wide phenotype-togenotype approaches have been employed by LD association mapping. An association mapping study employs the large number of recombination events that occur throughout the entire breeding selection history of the mapping population, thereby allowing fine-scale QTL mapping (Nordborg and Tavaré, 2002). Although the sample size is not sufficiently large in our association panel, the phenotypic variation in branch angle is very large. The heritability of this trait is relatively high, and it is related to significant genome loci with great effects. However, there must be existence of some unknown genes regulating branch angle in rapeseed comparing to model plant Arabidopsis and rice (Teichmann and Muhr, 2015), which still could not make of it. The novelty SNP clusters of QTL from our preliminary exploration are starting to decompose this aspect. Based on the MLM model, a total of 60 SNP associations (P < 2.90E −5 ) were detected for branch angle in three environments, and 25 significant SNP loci were further verified using BLUP model. Through the analysis of GWA, the markers detected in the environmental BLUPs value correlated significantly (P < 0.001) with branch angle, with phenotypic value effects between 16.60 and 18.93%. Exploring these associated markers provides a genetic basis to analyze branch angle variation in rapeseed.
The discovery of many false-positive QTL is due to the population structure (Zhao et al., 2007). To resolve this problem, several models have been developed including the Q+K model and the PCA model. Although the Q+K model have been demonstrated the most powerful method for identifying associations by many studies (Yu et al., 2005;Stich and Melchinger, 2009), we also compared different models and obtained the same conclusion that the MLM model (Q+K) was most suitable for our population. In our study, all B. napus accession lines can be largely divided into two subpopulations in STRUCTURE and compared with an association analysis from a previous study (Harper et al., 2012), and the population structure classification leads to the same results. Hence, the reasonable results for a population structureassociated analysis provide a foundation and guarantee. For the association analysis, we used a mixed model approach that avoided a confounding effect in the population structure and population relations. We acquired the branch angle BLUP values in three independent environments to eliminate the influence of environment.
To account for the potential number of false positives, we implemented stringent quality control on the included polymorphisms, with conservative non-parametric testing and an adjusted statistical significance threshold. Our approach relied on natural variations in rapeseed, leading to a set of strong candidate genes by comparative genome method. A high degree of co-linearity and congruence in the A. thaliana and Brassica genomes has been recognized (Parkin et al., 2005;Ziolkowski et al., 2006) and a single locus in the Arabidopsis genome is generally represented by three distinct loci in diploid Brassica species. Several genes that play key roles in regulating branch development in Arabidopsis have been identified. By applying this method to the recently grown tetraploid crop B. napus, we identified genomic regions that underlie four QTL for branch angle. The detected regions contained two Lazy (AT5G14090) orthologous, SPL14 (AT1G20980) and Auxin-responsive GH3 family protein (AT5G51470) genes, which were all relevant to angle formation in Arabidopsis thaliana.
Rapeseed is an excellent model for association analyses because of the extensive architectural variation across its native range and its diverse germplasm collections through artificial selection. During rapeseed domestication and breeding process, breeders often focus on important characteristics such as oil content, biotic stress resistance, and quality for directional selection for greater oil yield and better quality, which were directly caused by an artificial selection gene locus mutation at a lower frequency. For example, in terms of oil and glucosinolate contents, and because of the artificial selection on Fatty Acid Elongation 1 (FAE1) and High Aliphatic Glucosinolate 1 (HAG1) gene locus, gene locus mutation occurs at a lower frequency in double-low rapeseed populations . However, the optimation of rapeseed architecture (branch angle and plant height) to improve yield is more urgent nowadays. Associationmapping approach has advantage for distinguishing the most favorable alleles within a diverse genetic background, which provides the necessary genotypic information to facilitate the design of efficient rapeseed introgression and selection schemes throughout the world. This study indicated that the SNP variation frequency control of rapeseed branch angle habits is slightly higher than the results. This finding may be related to the domestication and genetic improvement of characteristics that are not particularly selected by the breeding program.
The association of branch angle with increased plant productivity in rapeseed may be beneficial to lodging resistance and high-density field cultivation. This genetic analysis of branch angle and plant morphological characteristics was only based on one natural population. Therefore, the observed inter-trait and trait-marker associations must be confirmed in other rapeseed populations. Further evaluations of branch angle and plant morphology should be conducted under contrasting cropping density, and total seed yield measurements should be taken because the seed harvest index in addition to the plant growth and productivity are strongly affected by the cropping density.

CONCLUSION
In this study, GWA mapping with corrections for the population structure were used to identify a number of novel loci and refine the map locations of known loci related to branch angle in rapeseed. This information not only demonstrates that GWAS mapping can be used as a powerful tool for dissecting plant architecture mechanisms in rapeseed but also provides valuable markers for breeding rapeseed cultivars with the ideotype. In addition, the candidate genes nearby these SNP loci represent promising targets for efforts to further identify causal variants and to clarify how the implicated genes affect branch angle in rapeseed.

AUTHOR CONTRIBUTIONS
JL and QH conceived and designed the study. JL, WW, and DL conducted the experiments; DM and HW coordinated genotyping with SNP markers; LF and YL provided rapeseed lines; JL and WW analyzed and interpreted data, and prepared the manuscript; QH supervised the whole study; all authors reviewed and edited the manuscript.


extfieldsofstudy: Chemistry Medicine

text:
Central Nervous Activity upon Systemic Salicylate Application in Animals with Kanamycin-Induced Hearing Loss - A Manganese-Enhanced MRI (MEMRI) Study

This study investigated the effect of systemic salicylate on central auditory and non-auditory structures in mice. Since cochlear hair cells are known to be one major target of salicylate, cochlear effects were reduced by using kanamycin to remove or impair hair cells. Neuronal brain activity was measured using the non-invasive manganese-enhanced magnetic resonance imaging technique. For all brain structures investigated, calcium-related neuronal activity was increased following systemic application of a sodium salicylate solution: probably due to neuronal hyperactivity. In addition, it was shown that the central effect of salicylate was not limited to the auditory system. A general alteration of calcium-related activity was indicated by an increase in manganese accumulation in the preoptic area of the anterior hypothalamus, as well as in the amygdala. The present data suggest that salicylate-induced activity changes in the auditory system differ from those shown in studies of noise trauma. Since salicylate action is reversible, central pharmacological effects of salicylate compared to those of (permanent) noise-induced hearing impairment and tinnitus might induce different pathophysiologies. These should therefore, be treated as different causes with the same symptoms.

Introduction
Salicylate is known to induce reversible loss of auditory sensitivity and tinnitus both in humans [1][2][3][4][5][6][7][8] and animals [9][10][11][12][13][14][15][16]. Therefore, the substance has been used in several animal studies that aimed to investigate the generation of tinnitus. Previous in-vivo and in-vitro studies have shown that acute application of sodium salicylate causes changes of outer (OHC) and inner (IHC) hair cell function, as well as an altered activity in central auditory and non-auditory structures. In the inner ear (the cochlea), salicylate leads to both functional and structural changes. Salicylate markedly reduces the cochlear blood flow by vasoconstriction [17]. Furthermore, changes in IHC synaptic morphology occur [18] with hair cell resting potential modified

Materials and Methods Animals
In the present study, 30 normal hearing adult (postnatal 30-60 day) mice (Mus musculus, NMRI strain) of both sex were examined. Different animals were used for threshold testing and MEMRI measurements. The experimental protocol was approved by the governmental commission for animal studies (LaGeSo Berlin, approval number: G 0153/06). Experiments were carried out in accordance with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes. All efforts were made to minimize the number of animals and their suffering. The condition of animals was monitored every 6 hours after the induction of hearing loss and, in addition, every hour following substance injection (kanamycin, salicylate or manganese, respectively). Since animals did not show any abnormal behaviour, additional analgesic treatment was not necessary.

Experimental induction of kanamycin-induced hair cell damage
For 22 mice, cochlear tissue was damaged by a subcutaneous injection of kanamycin sulfate (Carl Roth, Karlsruhe, Germany) dissolved in phosphate-buffered saline (PBS), at 1 mg/g body weight followed 40 minutes later by an intraperitoneal injection of bumetanide (Sigma-Aldrich, St. Louis, MO, USA) at 0.05 mg/g body weight. This procedure has been shown to elicit a significant and rapid hair cell loss in mice in-vivo [56]. Three of the above treatments were administered, each separated by 48 hours, to ensure a profound loss of sensory outer and, to a lesser extent, inner hair cells. Following kanamycin and bumetanide treatment, 7 animals underwent ABR recordings while 15 mice were used for MEMRI measurements.

ABR measurements
Auditory thresholds were measured for 7 hearing-impaired mice (48 hours after the final injection) and for 8 normal hearing control mice. Different animals were used for each group. The time interval for deaf mice was chosen to avoid any additional central pathophysiological effects arising from the damaged peripheral auditory system.
Frequency-specific (4,8,12,16 & 20 kHz) auditory brainstem responses (ABR) were recorded under anaesthesia. Sub-dermal needle electrodes were placed at the vertex (active), mastoid (reference) and at one foot (ground). Tone stimuli were delivered binaurally at different SPLs with a sine-wave generator (Modell SSU2, Werk für Fernmeldewesen, Berlin, Germany). ABR recordings were carried out using a Viking IV 1 measurement system (Viasys Healthcare, Conshohocken, Pennsylvania, USA). The brainstem responses (recording epoch 10 ms following stimulus presentation) were amplified (100.000 x), filtered (bandwidth 0.15-3 kHz) and averaged (300 x) by the Viking IV 1 . The amplitude growth function of wave IV/V was calculated from the resulting data for each frequency tested and a linear regression was fitted to the function. By extrapolating the regression line to zero, mean auditory thresholds were estimated for the controls and the kanamycin-treated animals. Results are shown as mean auditory thresholds (± S.E.) in dB SPL by group and mean relative hearing loss of the treated group compared to the normal hearing controls.

Salicylate application
To investigate salicylate-induced changes in central calcium-dependent activity after kanamycin-induced damage of cochlear hair cells, 9 treated animals received an intraperitoneal (i.p.) injection of a sodium salicylate (SS) solution (250 mg/kg). It has been shown earlier that salicylate is able to cross the blood brain barrier shortly after systemic (e.g. i.p.) application leading to an increased cerebrospinal fluid level of salicylate [57][58][59]. Comparable SS concentrations were also used in former studies to elicit salicylate-induced tinnitus in rodents [13,60,61]. SS was injected 48 hours after the last kanamycin/bumetanide treatment.

Manganese-enhanced magnetic resonance imaging (MEMRI)
MEMRI is a powerful tool to image central nervous system activity in small animals in-vivo [62][63][64]. After systemic application of a manganese chloride solution, manganese ions cross the blood-brain barrier [65] and enter excited cells by substituting calcium influx during neuronal activity [64,66,67]. Due to a slow clearance, Mn 2+ accumulates within the tissue which results in an increase in MRI-T1 signal contrast and therefore reflects the Ca 2+ -dependent activity [68]. Thereby, neuronal activity is monitored using the MEMRI technique and, thus, Ca 2+ -dependent activity can be imaged. This provides an opportunity to integrate neuronal activity, represented by the increase in signal contrast due to manganese accumulation, over a well-defined period of time before measurements, i.e., 24 hours in the present experiments. This is of particular importance during the investigation of auditory-related activity even inside a noisy MRI scanner [52,[69][70][71].
On the day of the experiments and 48 hours after the final kanamycin/bumetanide treatment (i.e. immediately after SS injection in the salicylate group), animals of the experimental group (treated with kanamycin/bumetanide and salicylate) as well as hearing-impaired control mice (kanamycin/bumetanide-treated only) received a 0.4 mM/kg dose of MnCl 2 solution (in accordance with [71]). Delivery was via a single intraperitoneal injection. Twenty-four hours after the manganese treatment (prior to this animals were kept in their cages in a quiet environment), when manganese accumulation reached its maximum level in the relevant brain structures [72], MRI scanning was performed. During MRI scanning, anaesthetised mice were placed on a heated circulating water blanket to ensure constant body temperature of 37°C. Anaesthesia was induced with 3% and maintained with 1.5-2.0% isoflurane (Forene, Abbot, Wiesbaden, Germany) delivered at 0.5 l ⁄ min of 100% O2 via a facemask under constant ventilation monitoring (Small Animal Monitoring & Gating System, SA Instruments, Stony Brook, New York, USA). Animals were euthanized after MRI measurements by an overdose of isoflurane (>5% isoflurane at 0.5 l/min of 100% O2).
Manganese-enhanced MRI was carried out using a 7 Tesla rodent scanner (Pharmascan 70 / 16AS, Bruker BioSpin, Ettlingen, Germany) with a 16 cm horizontal bore magnet and a 9 cm (inner diameter) shielded gradient with a H-resonance-frequency of 300 MHz and a maximum gradient strength of 300 mT/m. For imaging a 1H-RF quadrature-volume resonator with an inner diameter of 20 mm and a T1-weighted 2D turbo spin-echo sequence (TR / TE = 938 / 10.6 ms, RARE factor 2, 6 averages) was used. Data acquisition and image processing were conducted using the Bruker Paravision 4.0 software. Thirty-five axial slices with a slice thickness of 0.3 mm, a field of view of 2.85 x 2.85 cm and a matrix of 256 x 256 resulted in an in-plane resolution of 111μm. Imaging covered the brain from brainstem to forebrain. Signal intensity analysis of defined auditory brain regions was carried out with the program Analyze 5.0 (AnalyzeDirect, Inc.; Lenexa USA). Signal intensities were quantified bilaterally in the dorsal (DCN) and ventral (VCN) cochlear nucleus, the superior olivary complex (SOC), the inferior colliculus (IC), the medial geniculate body (MGB), the auditory cortex (AC), the preoptic area of the anterior hypothalamus (PO/AH), the Amygdala (Amyg). The masseter muscle was used as an objective intensity reference, since it is located close to the related neural tissue but is not affected by changes in brain activity. PO/AH and Amyg served as non-auditory structures investigated in the present study. Regions of interest were marked in accordance with "the mouse brain atlas in stereotaxic coordinates" [73] (Fig 1). The investigated brain structures were manually outlined within each relevant brain slice and a voxel-based analysis was carried out for each structure under observation. Due to variations in each animal's position within the MRI scanner, the dimensions of each brain structure (the tissue volume) in the scan could vary across subjects. Therefore, the number of voxels included in each structure differs slightly. Signal intensities for all analysed structures were normalized in relation to the intensity of the muscle at the same side. The relative MRI signal (given in normalized relative units) was calculated for each animal by using the mean of the measured signal strength of every slice for each analysed structure, normalized in relation to the intensity for the muscle at that side. The results of all animals were compared between the salicylate group and the hearing-impaired control group for each investigated brain area.

Statistical analysis
The statistical comparison was done with the software SPSS (IBM SPSS Statistics Version 20, IBM Corp., Armonk, New York, USA). After confirming a normal distribution using the Kolmogoroff-Smirnoff-test, the t-test for independent samples was applied. A statistically significant difference was considered for p<0.05.

ABR-recordings
Hearing loss following kanamycin and bumetanide treatment was indicated by an ABR-threshold shift of up to 40 dB in the investigated frequency range between 4 and 20 kHz compared to the untreated normal hearing control group. Frequency-dependent mean auditory thresholds in normal hearing controls ranged between 17 and 31 dB SPL. In the hearing impaired animals, thresholds were detected between 53 and 60 dB SPL (Fig 2). Average hearing loss in the experimental group ranged between 26 dB at 4 kHz and 40 dB at 16 kHz. However, threshold shift was highly significant for all frequencies (p<0.001).

MEMRI
Following systemic salicylate application, MEMRI contrast increased significantly in all investigated auditory brain areas.
An increased MEMRI signal was also observed for the non-auditory control areas (PO/AH and Amyg) after salicylate treatment. Signal intensities were elevated from 148.5±2.0 (hearing-impaired controls) to 175.0±2.1 (salicylate group) in PO/AH and from 143.6±3.0 in hearing-impaired controls to 167.8±2.5 in salicylate-treated animals in the amygdala region (Fig 3). All data are given in normalized relative units (calculated from the measured signal intensities, as described in the methods section) as mean ± S.E.
The differences between the groups were statistically significant for all investigated auditory and non-auditory structures (p0.001 for all comparisons).

Discussion
The results of the present study demonstrate changes in calcium-dependent brain activity after systemic salicylate application in kanamycin-treated animals using non-invasive manganeseenhanced magnetic resonance imaging (MEMRI).

ABR thresholds
The ABR recordings demonstrated that a three-fold application of kanamycin and bumetanide produces a significant threshold shift in mice, possibly in particular due to OHC destruction according to previous reports. It is further assumed that the treatment induces a minor loss of IHC and spiral ganglion cells as well [56,74]. Although it has not been directly investigated here, such a decrease of sensory cells is supported by the electrophysiological findings presented here. The hearing loss after kanamycin and bumetanide treatment of up to 40 dB matches to the results of other studies showing that a near-complete loss of OHC function in mice results in a comparable shift of auditory thresholds [26]. These findings lead to the assumption that the observed central effects of salicylate are only marginally related to its action on outer hair cells. Inner hair cells might also be affected which could further contribute to our results.

Ca 2+ -related effect of salicylate after peripheral hearing impairment
Recent studies have reported that the central auditory system largely contributes to the salicylate-induced effects on neuronal activity and auditory phantom perceptions like tinnitus, even after disconnection of the auditory periphery [54,75,76]. In the present data, the calciumrelated neuronal activity was increased after systemic application of a sodium salicylate solution in several investigated brain structures. This followed kanamycin-induced reduction of the peripheral input from cochlear sensory cells. In addition, it was shown that the central effect of salicylate is not limited to the auditory system, but shows a rather general alteration of calcium-related activity, as indicated by the increased manganese accumulation in the preoptic area of the anterior hypothalamus (PO/AH) as well as in the amygdala. Basta et al. [55] showed an increased spontaneous activity during salicylate superfusion in brain slices of auditory and non-auditory (hypothalamic) structures. The hypothalamus is a central structure responsible for thermoregulation, which maintains the body temperature at a constant level. When salicylate is given in patients with elevated temperature (fever), it exerts an antipyretic effect. In rats, the intra-ventricular application of salicylate close to the PO/AH can induce hypothermia [77], which might be related to an altered neuronal activity in the corresponding structure as shown in the data (body temperature was kept constant during MRI scanning only). This additional activity increase, despite the overall changes in neural tissue, could also provide an explanation for the higher salicylate-induced effect in the hypothalamus and amygdala compared to the auditory system. Further support comes from earlier findings that systemic application of salicylate leads to a strong decline in body temperature [78].
An altered activity of the amygdala after salicylate application has already been reported by immunostaining. An elevated c-Fos-or arg3.1-related activity was demonstrated in the auditory cortex and the amygdala after treatment with a single high-dose salicylate application [33], whereby a decrease was observed in VCN and IC. In the present study, an increase in MEMRI signal intensity within the amygdala and therefore, an elevation in calcium-related activity, might rely on similar mechanisms responsible for synaptic transmission. However, further investigation is needed to clarify the differences in subcortical auditory structures.
One possible mechanism for this increase in calcium-related activity could be the direct action of salicylate on neuronal and synaptic activity within those particular brain structures. Salicylate largely influences the pre-and postsynaptic conductance of several transmembrane ion channels by modulating neurotransmitter activity (e.g. modulation of GABA-mediated inhibition) [38,40]. In turn, the activation of neurons by releasing action potentials and increasing excitatory synaptic transmission can be induced [37,[79][80][81]. Further, an increase in immediate early gene activity and its possible function in upregulation of NMDA receptor activity was demonstrated in the dorsal cochlear nucleus [41]. These mechanisms depend on intracellular calcium and might induce calcium and manganese influx into the cell, both being able to elicit several calcium-related neuronal responses [65,66,82]. This might have contributed to an elevated intracellular manganese accumulation followed by a higher MEMRI signal intensity in the present data. This idea is supported by our recent findings of salicylate effects in brain slices [55].
It should be mentioned that pharmacological removal of cochlear sensory cells reduces auditory nerve input towards the central auditory system. This happens particularly when inner hair cells have been affected (in addition to OHC damage), thereby inducing changes in central neuronal activity (e.g. compensatory hyperactivity), as shown by several studies in this field. For a review, see [83]. However, hearing impaired controls as well as salicylate-treated animal should be affected to a similar extent. Another important issue is that salicylate application itself has the property to further diminish sensory function. It has been reported that salicylate particularly influences outer hair cell motility and thereby significantly reduces stimulus-driven auditory nerve activity [22,46,84,85]. Any such effect should play only a minor role in the present data, since spontaneous calcium-related activity was measured and animals were kept in a quiet environment to prevent external acoustic stimulation during the experiments. However, with the current experimental design, an additional salicylate-induced hearing impairment and a further salicylate-driven effect on the function of surviving hair cells could not be excluded completely and might therefore slightly contribute to the observed effects.
The present study indicates that salicylate-induced activity changes in central auditory brain areas, including possibly tinnitus, exist to a large extent. Since observations were made after reduction of peripheral effects mediated by cochlear hair cells, these effects cannot be entirely explained by salicylate's action on peripheral tissue.

Is there a correlation with noise-induced hearing loss/tinnitus?
The present results indicate that the mechanisms through which salicylate impacts on activity in several auditory and non-auditory brain structures appears different to those known from noise-induced hearing loss (and possibly tinnitus generation); particularly immediately after treatment. Acute noise-exposure is accompanied initially by cochlear changes. In a second stage, the altered afferents are probably responsible for modulating central auditory processing. However, permanent tonotopic and other pathophysiological changes appear to be largely established by compensatory neuroplasticity, acting to overcome the reduced cochlear output and damaged central auditory structures. This is in line with the finding that an increased spontaneous firing rate (SFR) in the DCN (after 140 dB SPL noise exposure) could be recorded only after a 2-3 days' period [86]. Once these changes are established as autonomous nervous activity, they seem to become uncoupled from cochlear projections and show a rebalancing of central inhibitory and excitatory neurotransmission [87,88]. It has been demonstrated that sectioning of the auditory nerve and, thus, abolishing the peripheral input, had no effect at all on the increased SFR in the DCN after noise exposure [89]. Further discrepancies between the impact of noise and salicylate became apparent when comparing the current data with our recent findings using the MEMRI technique. In earlier MEMRI experiments we demonstrated that noise-induced hearing loss leads to short-and long-term changes in calcium-related activity in central auditory structures [70]. As the generation of tinnitus was not tested in both studies, differences in Ca 2+ activity patterns between the two studies even indicate that hearing impairment related to either salicylate application or noise trauma have varying impacts on central auditory system physiology and might therefore, rely on different pathophysiological mechanisms. This may lead to the hypothesis that the appearance of tinnitus due to salicylate or noise is also based on different sources. Calcium-related activity patterns have also been observed by another study investigating activity changes in several brain structures after induction of noise-or salicylate-related tinnitus using manganese-enhanced MRI [52]. Signal intensity differences between noise and salicylate treatment were only present in some of the structures investigated: e.g. the DCN and part of the inferior colliculus (DCIC). The discrepancies between these studies might be related to the particular experimental design, as Holt and colleagues used rats and their animals were normal hearing before treatment. Moreover, noise was delivered unilaterally and was narrow-band, which is in contrast to our recent work and could explain the different results.
In essence, the present data support the idea that the impact of salicylate application or noise trauma on the auditory system are probably based on different mechanisms, even if comparable changes of electrophysiological properties could be observed in vivo: e.g. increased SFR in central auditory areas [47,90,91], alterations in synaptic transmission [9,40,41,92,93] and an increase in neural synchrony [51,94]. Since salicylate action is reversible, central pharmacological effects of salicylate compared to those of (permanent) noise-induced hearing impairment and tinnitus seem to induce different pathophysiologies and might therefore, be considered as different causes for the same symptoms.
Supporting Information S1