Such multi-parametric approach is expected to better describe maturational asynchrony than conventional univariate approaches because it takes into account complementary dependencies of the parameters on different maturational processes, notably the decrease in water content and the myelination. Our approach was tested on 17 healthy infants (aged 3- to 21-week old) for 18 different bundles. It finely confirmed maturational asynchrony across the bundles: the spino-thalamic tract, the optic radiations, the cortico-spinal tract and the fornix have the most advanced maturation, while the superior longitudinal and arcuate fasciculi, the anterior limb of the internal capsule and the external capsule have the most delayed maturation. Furthermore, this approach was more reliable than univariate approaches as it revealed more maturational relationships between the bundles and did not violate a priori assumptions on the temporal order of the bundle maturation. Mahalanobis distances decreased exponentially with age in all bundles, with the only difference between them explained by different onsets of maturation. Estimation of these relative delays confirmed that the most dramatic changes occur during the first post-natal year.In vivo evaluation of the brain white matter maturation is still a challenging task with no existing gold standards. In this article we propose an original approach to evaluate the early maturation of the white matter bundles, which is based on comparison of infant and adult groups using the Mahalanobis distance computed from four complementary MRI parameters: quantitative qT1 and qT2 relaxation times, longitudinal λThe online version of this article (doi:10.1007/s00429-014-0881-y) contains supplementary material, which is available to authorized users. In addition to ordering the bundles according to their relative maturation, our approach suggests a general description of the maturation that allows estimating the relative maturational delays between the bundles.According to all these constraints, we introduce here a novel strategy to reliably describe and efficiently compare the bundles maturation in infants from 1 to 5 months of age. This strategy is based on estimation of the Mahalanobis distance between the multi-parametric vectors of four parameters , with a maturational age  between 3 and 21 weeks. Infants were compared to an adult group of 13 healthy subjects . Additionally, a 34-week-old girl  was imaged for the model evaluation at an older age. None of the subjects displayed any neurodevelopmental problems or any brain abnormalities observed on MR images. The study protocol was approved by the regional ethical committee for biomedical research; all parents and adult subjects gave written informed consents. Infants were spontaneously asleep during MR imaging. Particular precautions were taken to minimize noise exposure, by using customized headphones and covering the magnet tunnel with special noise protection foam.2, matrix = 128 × 128) using EPI single-shot spin-echo (SE) sequences . For DTI, a DW-SE-EPI sequence was used with 30 orientations of diffusion gradients with b = 700 s mm−2 (+b = 0 volume): TE = 72 ms, TR = 10 s (TR = 14 s for adults), parallel imaging GRAPPA factor 2, partial Fourier sampling factor 6/8, leading to an acquisition time of 5 min 40 s (7 min 56 s for adults). For qT1 mapping, an inversion recovery (IR) SE-EPI sequence was used with eight different values of inversion time : TE = 38 ms, TR = TI + 15 s (TR = TI + 21 s for adults), partial Fourier sampling factor 5/8, leading to an acquisition time of 2 min 11 s (3 min 03 s for adults). For qT2 mapping, an SE-EPI sequence was used with 8 different values of echo time (TE = 50→260 ms each step 30 ms): TR = 15.5 s (TR = 21.7 s for adults), parallel imaging GRAPPA factor 2, partial Fourier sampling factor 6/8, leading to an acquisition time of 2 min 51 s (4 min for adults).Data acquisition was performed on a 3T MRI system , equipped with a whole body gradient  and a 32-channel head coil. Interleaved axial slices covering the whole brain were imaged with a 1.8-mm isotropic spatial resolution  using Connectomist software , spino-thalamic tract STT, optic radiations OR, anterior limb of the internal capsule ALIC;association bundles: external capsule EC, arcuate fasciculus AF, superior SLF and inferior ILF longitudinal fascicles, uncinate fasciculus UF, fronto-occipital fasciculus FOF;limbic bundles: fornix FX, inferior CGinf and superior CGsup parts of the cingulum;commissural bundles: genu CCg, body CCb and splenium CCs of the corpus callosum.After correction of artifacts from motion and eddy currents .For all bundles, comparison of the normalized parameters in the infant and adult groups was performed using Mahalanobis distance Possible bias from non-dominant components that appears in small samples was compensated by substituting the smaller eigenvalues with the maximal eigenvalue  then the bundles were said to have different maturational trajectories. The result of the pair-wise comparisons between all bundles created a partial maturational order on the set of bundles that was presented as a graph, showing complex maturational relationships. Statistical tests were considered with a 0.95 significance level, corrected for multiple comparisons using FDR approach. Relationships that failed to reach the 0.95 significance level were also tested at the level of 0.9.To compare two bundles across the whole age range, these differences were considered between the corresponding age-points on their maturational trajectories. If these differences were significantly different from zero  were compared in terms of (1) the number of discriminated relationships between the bundles; (2) presence of violations in five a priori known maturational relationships: spino-thalamic tract, cortico-spinal tract and optic radiations should be among the most fast-maturing bundles, while anterior limb of the internal capsule and arcuate fasciculus should be among the most slowly maturing (Yakovlev and Lecours R2 > 0.46 corresponding to p < 0.05). To make predictions for each bundle, the fitting was done using all but one infant, and his/her age predicted by the fitting was then compared with the real age. The described procedure was repeated for all infants and the prediction errors were averaged.Additionally, we evaluated which strategy made better predictions on the maturational age using a “leave-one-out” approach. Because of the short age range, changes in the normalized MRI parameters and in the Mahalanobis distance with age were fitted with linear equations . For each parameter, the observed differences across the bundles suggested that certain bundles  matured faster than the others; however, the majority of the bundles could not be differentiated one from another.Besides, Mahalanobis distance was computed for each bundle in all infants: it decreased with age in all bundles, reflecting bundles’ maturation Fig. a. It seePair-wise comparisons of Mahalanobis distances across the bundles created a partial maturational order represented as a graph Fig. b. As exp⊥). Thus, according to our comparison criteria, Mahalanobis distance showed the best performance . Thus, the maturational trajectories were further modeled by exponential decays  and longitudinal (λ║) diffusivities. Fractional anisotropy (FA) and mean diffusivity (<D>) were not considered because they can be viewed as functions of λ⊥ and λ║, and including them may result in a degenerate covariation matrix.Our approach is free from these drawbacks, and to our knowledge, it is the first study using Mahalanobis distance to evaluate brain maturation. Mahalanobis, rather than Euclidean distance, was chosen because MRI parameters are correlated and cannot be viewed as completely independent variables. Moreover, their covariation matrices and thus, the eigensystems are different across bundles. In our study, Mahalanobis distance was calculated using four parameters: quantitative relaxation times , transverse . Besides, it might also be possible that co-registration of the different imaging modalities  in the lower parts of the brain was not as perfect as in the central regions (because geometric distortions related to EPI sequences are more prominent in the brainstem) and the relative maturational degree was slightly underestimated in the inferior portion of the cortico-spinal tract.Next, we should also highlight that the Mahalanobis distance is not directly linked to the myelin content but rather reflects the whole ensemble of various maturational processes underlying age-related changes in the MRI/DTI parameters used for its calculation  included in this work may seem relatively low to derive definite conclusions about white matter maturation, particularly for the white matter bundles showing higher prediction errors in the leave-one-out validation , i.e. the bundles in which inter-subject variability in the MRI/DTI parameters or in the Mahalanobis distance was relatively high as compared to the age-related changes over the considered age period. Nevertheless, as most of the bundles indeed showed dramatic changes of both the Mahalanobis distance and the MRI/DTI parameters over this short developmental period (3–21 weeks), we were able to reveal a general scheme of the maturational asynchrony across the bundles, even in a cross-sectional analysis. However, the main goal of the present study was not to make definite conclusions about the exact bundle maturational order, but rather to introduce and explore the Mahalanobis distance approach and to demonstrate its advantages over conventional univariate approaches. Indeed, even in a small size group, Mahalanobis distance approach showed better performance than conventional approaches in bundle discrimination and suggested a more reliable bundle ordering with smaller prediction errors of the maturational age. Although the maturational order may be considered here as preliminary, requiring further validation in studies with larger sample sizes, notably to distinguish the bundles that were grouped together (see above), the obtained results suggest that our approach may be a promising candidate for the evaluation of pathological development or neuro-degeneration of the white matter when it is not possible to acquire large datasets. Similarly, interpolation of our model to older ages should be made with caution, since it was only tested in a single 34-week-old infant for demonstration purposes. Testing whether the exponential model and the Mahalanobis approach are indeed valid at older ages would require recruiting many healthy infants and toddlers during the second semester of the first post-natal year and the first semester of the second year . This is hardly achievable because it is exceptional to have healthy infants and toddlers spontaneously asleep (without sedation) during scanning at those ages.The number of infants   or when the patients cannot withstand long acquisitions (e.g. psychiatric patients).Supplementary material 1 (PDF 126 kb)Supplementary material 2 (PDF 167 kb)Below is the link to the electronic supplementary material.


Allergic rhinitis (AR) is characterised by burdensome nasal and/or ocular symptoms. This inflammatory disease can be debilitating and thus result in considerable health-related and economic consequences.In a cross-sectional study, adult subjects with AR (N = 683) completed three allergy-specific questionnaires that assessed the impact of AR on the work/academic performance, daily activities, health-related quality of life (HRQOL), and satisfaction with allergen immunotherapy (AIT). Regression analyses were used to examine the associations between several clinical variables and the patient-reported outcomes.Total loss of productivity was 21.0 and 21.2 % for employed and student patients, respectively, whereas the impairment of daily activities was 22.0 %. The mean overall HRQOL score was 1.94 ± 1.29 . Global score for satisfaction with AIT was 65.5 ± 24.8 . Simple regression analysis found statistically significant associations between loss of work and academic productivity, impairment of daily activities and the type and severity of AR. AIT was a protective factor. The persistent and more severe types of AR and lack of AIT contributed to the worsening of HRQOL.AR (the persistent and more severe form of the disease) has an impact on functional characteristics of adult patients in Spain. AIT might reduce the effect of this disease on the work/academic performance and HRQOL.Trial registration Retrospectively registered Allergic rhinitis (AR) affects more than 400 million people worldwide, with high prevalence recorded in the developed countries , 2. It iAR is also associated with substantial economic costs , 10. IndIn Spain, a previous study estimated direct and indirect costs of AR, showing the important economic burden of the disease . The worThe study was a cross-sectional, observational study conducted in allergy departments in Spain from May 2011 to October 2012. The study was approved by the Institutional Ethics Committee of the Hospital German Trias i Pujol  and was conducted in accordance with the principles of the Declaration of Helsinki. Before participation, all patients had signed the informed consent form.A number of physicians from different regions were selected to cover the geographical area of Spain Fig. . Each phThe study data collection was performed during a single visit. Physicians completed a patient record form for each patient. The record included demographic characteristics, the information on the years since diagnosis of the disease, allergens/factors inducing nasal symptoms, disease severity, number of visits to the specialist in the last 12 months, symptoms and comorbidities , symptomatic treatment for AR, and the use of allergen immunotherapy (AIT).Patients were invited to complete several self-report questionnaires. To evaluate the burden of illness, the Work Productivity and Activity Impairment Questionnaire and Classroom Impairment Questions: Allergy Specific (WPAI + CIQ:AS) were used . The WPAThe HRQOL assessment was performed using the ESPRINT-15 tool, the short form of the Spanish questionnaire specifically designed for AR patients , 19. ThiThe Satisfaction Scale for Patients Receiving Allergen Immunotherapy  questionnaire  was usedWe performed data management and statistical analysis using the SPSS software package . A descriptive analysis was conducted, describing categorical variables using numbers and percentages, and quantitative variables using means and standard deviation (SD).For the association of quantitative variables, the Kruskal–Wallis and Mann–Whitney tests were used. We determined predictive factors for impaired productivity and quality of life by using linear regression analysis. The dependent variables were the scores in the patient questionnaires. The multivariable model was developed, based on a backwards selection from the variables with significance in bivariate analysis. All statistical tests were considered significant at P < 0.05.The study enrolled 683 patients recruited by 144 physicians. The patient demographics are shown in Table Clinical information is presented in Table Mean WPAI + CIQ:AS scores were similar for the employed and the students, with the loss of productivity of 21.0 and 21.2 %, respectively. Overall mean value for the impairment of daily life activities was 22.0 %. The mean overall HRQOL score assessed using the ESPRINT-15 tool was 1.94 ± 1.29 . The symptom dimension had a high score (2.21 ± 1.38), whereas daily activities were less affected (1.67 ± 1.47). The global score in the ESPIA questionnaire filled by the patients under AIT (n = 508) was 65.5 ± 24.8 . Overall satisfaction with the treatment had the highest score (73.4 ± 25.7), while the score for the activity and environment dimension was the lowest (61.8 ± 27.3).Initially in the bivariate analysis, several clinical parameters were identified as potential factors significantly associated with impaired work/academic productivity and daily activities Table : age, seSimple regression analysis confirmed that work productivity was associated with the education level, with more education associated with less disease impact, type and severity of AR (persistent and more severe AR associated with more impairment), and current AIT (less impact with AIT). Academic productivity was associated with age (older people had lower impaiment), type of AR (persistent AR affected more), and current AIT (less impact with AIT). Impact on daily activities was associated with sex , type and severity of AR (persistent and severe forms had a greater impact), and current AIT (less with AIT). As shown in Table For the ESPIA scores, employed patients with persistent and more severe AR were less satisfied with their AIT, while being allergic to both dust mite and pollen and suffering intermittent AR were associated with higher scores in student patients. There was no significant difference between satisfaction with SCIT and sublingual immunotherapy (SLIT) Table .Table 5FFor the first time in Spain and under “real life” conditions, the ENERGY study examined the impact of AR on work/academic performance and HRQOL of adult patients poorly controlled with or without pharmacological treatment. The study used the validated questionnaires WPAI + CIQ-AS, ESPRINT-15, and ESPIA, the tools specifically developed for allergies. Our results suggest that AR affects several functional areas. The data are in accord with the existing reports of the impairment of work/academic productivity , 4, 6 anThe WPAI questionnaire has been used before to quantify the impact of several pathologies, such as irritable bowel disease, Crohn’s disease, and ankylosing spondylitis, on various aspects of productivity –24. HereImpairment in HRQOL of AR patients has been a frequent object of study , 30. DisThese results justify the effort put into optimizing the allergy therapies. Pharmacological treatments are generally effective and well tolerated, improve sleep quality, reduce daytime fatigue , 34, andOur results showed a negative impact of AR on work/academic productivity and HRQOL of patients receiving pharmacological treatment in several allergy departments in Spain. Several factors such as persistent AR or a more severe form of the disease were associated with higher impairment of the studied functional outcomes. Specific AIT may play a protective role, improving productivity and HRQOL of AR patients. A more comprehensive interaction between patient and physician might be required to reduce the current socioeconomic burden of this disease. Various parameters including nature, severity, and impact of AR symptoms on self-perceived health-related measures should be taken into account.


The aim of this study was to investigate the effects of curcumin on epididy-mal sperm parameters in adult male Navel Medical Research Institute (NMRI) mice ex-posed to sodium arsenite.In this experimental study, we divided the animals into fourgroups: control, sodium arsenite (5 mg/kg), curcumin (100 mg/kg) and curcumin+sodiumarsenite. Exposures were performed by intraperitoneal injections for a 5-week period.After the exposure period, we recorded the animals’ body and left testes weights. The leftcaudal epididymis was used to count the sperm number and analyze motility, viability,morphological abnormalities, acrosome reaction, DNA integrity, and histone-protaminereplacement in the spermatozoa. One-way analysis of variance (ANOVA) followed bythe Tukey’s test was used to assess the statistical significance of the data with SPSS 16.0.P<0.05 was considered significant.Mice exposed to sodium arsenite showed a significant decrease in the num-ber, motility, viability, normal sperm morphology and acrosome integrity of spermato-zoa compared to the control group. In the curcumin+sodium arsenite group, curcuminsignificantly reversed these adverse effects to the point where they approximated thecontrol. In addition, the application of curcumin alone had no significant differencein these parameters compared to the control and curcumin+sodium arsenite groups.However, we observed no significant differences in the body and the testis weight aswell as the DNA integrity and histone-protamine replacement in the spermatozoa ofthe four groups.Curcumin compensated for the toxic effects of sodium arsenite on a numberof sperm parameters in adult mice. Numerous evidence exists that today’s men have considerably lower sperm counts compared to those who lived 50 years ago . AspectsOxidative stress is associated with risk factors for infertility. Therefore, numerous studies have attempted to ameliorate the adverse effects of oxidative stress using antioxidant therapy to improve the endogenous antioxidant defense system. In this regard, application of antioxidants derived from plants can be an effective strategy for alleviating infertility caused by oxidative stress. Curcumin, a yellow phenolic pigment and active component of the rhizomes of Curcuma longa (turmeric), has a wide range of pharmacological and biological properties that include anti-inflammatory and anticancer effects ,12. CurcPrevious studies reported adverse effects of sodium arsenite on the male reproductive tract ,6. To our knowledge, however, no study examined the effects of curcumin on sodium arsenite mediated toxicity in epididymal spermatozoa of adult mice. The present study investigated the effects of curcumin on epididimal sperm parameters in adult mice exposed to sodium arsenite. libitum. Adult mice (n=24) were randomly divided into four groups: control, sodium arsenite   mice  were purchased from Pasteur Institute, Tehran, Iran. The animals were housed in plastic cages on a 12-hour light/dark cycle, temperature of 24 ± 2ºC, with water and food ad ma, USA) , curcumima, USA) ,23 and cThe dissected epididymis from each animal was transferred into 10 ml Ham’s F10 medium and cut into small slices in order to release the spermatozoa into the medium. After 10 minutes, one ml of the sperm suspension was diluted with 9 ml of formaldehyde. The diluted spermatozoa were transferred into a Neubauer hemocytometer chamber and the sperm heads were counted with a microscope. The sperm count was performed according to the World Health Organization (WHO) guidelines  and dataAssessment of sperm motility was performed according to a WHO protocol . BrieflySperm viability was evaluated by eosin-nigrosin staining according to a WHO protocol . In brieThe Diff Quick staining kit  was used to evaluate sperm morphology . The speThe ability of spermatozoa to undergo acrosome reaction was evaluated by the Coomassie brilliant blue staining method . The speIn order to investigate sperm chromatin structure in epididymal sperm, acridine orange (AO) staining was used to detect DNA integrity (double strand DNA versus single strand DNA) in the sperm. Aniline blue (AB) staining was performed to detect histone-protamine replacement during the sperm maturation process. AO staining was performed according to the Tejada et al.  method. The AB staining was carried out based on the Wong et al.  method. Results were expressed as mean ± SD for six animals per group. One-way analysis of variance (ANOVA) followed by the Tukey’s test was used to assess the statistical significance of data using SPSS  P<0.05 was considered significant. We observed no significant differences in body and testis weights among the four groups P>0.05, . There was significantly lower sperm count in the sodium arsenite group compared to the control group (P<0.01). In the curcumin+sodium arsenite group, curcumin significantly compensated the sperm count compared to the sodium arsenite group (P<0.01). Animals exposed with curcumin alone showed no significant difference in this parameter compared to the control and curcumin+sodium arsenite groups . Sodium arsenite significantly decreased the percentage of PMS (P<0.001) and increased the percentage of NPMS (P<0.01) as well as NMS (P<0.001) compared to the control group. In the group of animals exposed to curcumin+sodium arsenite, curcumine significantly (P<0.001) ameliorated the adverse effect of sodium arsenite on these motility patterns compared to the sodium arsenite group. There were no significant differences in sperm motility in the curcumin group compared to the control and curcumin+sodium arsenite groups . Sperm viability significantly (P<0.001) decreased in the sodium arsenite group compared to the control group. Administration of curcumin significantly reversed sperm viability in the curcumin+sodium arsenite group when compared to the sodium arsenite group (P<0.001). However, the application of curcumin alone had no significant effect on sperm viability compared to the control and curcumin+sodium arsenite groups . This table shows that the sodium arsenite induced a significant (P<0.01) increase in abnormal sperm. In the curcumin+sodium arsenite group, curcumin significantly reversed the percentage of sperm morphological abnormalities compared to the sodium arsenite group (P<0.01). Figure 1 shows a number of sperm abnormalities induced in sodium arsenite mice. Curcumin showed no significant effect on this parameter compared to the control and curcumin+sodium arsenite groups . In the sodium arsenite group there was a significantly lower percentage of spermatozoa with intact acrosome (P<0.001) compared to the control. Curcumin significantly (P<0.001) compensated the adverse effects of sodium arsenite on acrosomal reaction in the curcumin+sodium arsenite group compared to the sodium arsenite group. In addition, we observed no significant differencein acrosome integrity between the curcumin and control as well as curcumin+sodium arsenite groups . Interestingly, in all sperm parameters where curcumin in the curcumin+sodium arsenite group significantly reversed the toxic effect of sodium arsenite, the data mean was similar to that of the control group . Both AO and AB staining showed that neither sodium arsenite nor curcumin had any obvious effects on sperm DNA integrity and histone-protamine replacement . This study evaluated the spermatotoxic effects of sodium arsenite in adult mice and showed that curcumin had the capability to reverse sodium arsenite toxicity. In the present study, sodium arsenite had no significant effects on body and testis weights. Our results supported those of previous studies ,30, howeIn accordance with previous findings ,6, sodiuThe results of the present study showed a significant decrease in sperm motility and viability in the sodium arsenite group compared to the control group. Arsenic with its electrophilic nature has been shown to readily interact with thiol and sulfhydryl groups on proteins . The decThis study has explored the toxic effect of sodium arsenite on sperm morphology. Arsenic can cause changes in pituitary-gonadal axis hormones . TherefoWe showed that sodium arsenite significantly increased abnormal acrosome reaction in spermatozoa. Oxidative stress, by inducing lipid peroxidation, affected both fluidity and flexibility of spermatozoa membrane . The incPrevious studies reported DNA damage induced by ROS . AntioxiCurcumin, as a major component of turmeric and a natural antioxidant, is not toxic. Its adverse effect on the fetus has not been reported. Possibly, curcumin can be used in human diets as a therapeutic agent against different pathological conditions induced by oxidative stress.


Linum spp.) have been used historically as models for the study of SAMs  is grown in more than 50 countries for its seeds or its stem phloem (bast) fibers , which acts as a reservoir of stem cells. Members of the flax genus . In total, 117.5 million clean reads (21 Gbp) were obtained and mapped to the reference flax genome to detect transcripts enriched in the AR as compared to the BR. These differential transcript expression data  are available at NCBI GEO , and an annotated version of the file is available as Supplemental Table We used RNAseq to compare transcript expression patterns in two segments of the vegetative stem of 14d flax plants, from which all visible leaves had been removed. The segments were: (i) the apical region (AR) of the shoot apex, which contained the apical-most 0.5 mm of the stem, including the SAM and its immediate derivatives; and (ii) the basal region (BR), which contained the entire stem except for the apical-most 1 cm, and therefore represented all stem and vascular tissues at later stages of differentiation as compared to the AR. Four biologically independent replicates of AR , and two biologically independent replicates of BR  were sequenced on the Illumina HiSeq platform in a total of nine runs. Data were deposited in the Sequence Read Archive (SRA) as the following accessions: AR1: L. usitatissimum L. cv. CDC Bethune; Rowland et al., Flax  plants (E. coli DNA PolI (Invitrogen). Double-stranded cDNA , was sheared with a nebulizer, end repaired, and ligated to Illumina PE adapter oligos, and the products size-selected by gel purification to produce 200 bp fragments. These were PCR amplified through 15 cycles to prior to sequencing using an Illumina HiSeq 2000 with 90 bp, paired-end reads. The quality of the sample during processing prior to sequencing was monitored using the Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System. Because the sequencing output for samples AR2, AR3, and AR4 was slightly lower than expected (9.6 million reads output per sample), additional aliquots of each of these three samples were sequenced in three additional runs. Raw reads from all runs were filtered to remove adapter sequences, contamination, and low-quality reads, and the filtered raw reads were deposited in the SRA archive. Each of the nine paired read files were uploaded to SRA in fastq format.RNA from each biological replicate  was extracted separately. RNeasy Micro Kit (Qiagen) and RNeasy Plant Mini Kit (Qiagen) were used to isolate RNA from the AR and BR samples, respectively. Extracted RNA was then digested with TURBO DNA-free™ Kit (Life Technologies) to remove DNA contamination and their quality was evaluated using a RNA 6000 Nano chip (Agilent Technologies) on an Agilent 2100 Bioanalyzer. Total RNA was delivered to the service provider, BGI , where each biological replicate was sequenced separately. oligodT coupled magnetic beads were used to isolate poly-A+ mRNA, which was used as a template for cDNA synthesis  primed by random hexamers, followed by second strand synthesis using GSE80718, and an annotated version of this file is available as Supplemental Table q < 0.05) more abundant in AR compared to BR, and 4405 of these were enriched at least 2-fold in the AR. Conversely, transcripts for 8388 genes were significantly (q < 0.05) more abundant in BR compared to AR and 7901 of these were enriched at least 2-fold in the BR. Inspection of the data showed that several markers of shoot apex tissues were highly enriched in the AR sample. For example, PROTODERMAL FACTOR 1 (PDF1) transcripts have been reported to be expressed exclusively in the L1 layer of meristems and the protoderm of organ primordia  were at least 19.5-fold more abundant in AR than BR  genes are required for SAM function and organ separation  were at least 45-fold more abundant in AR than BR; two other putative CUC genes  were not detected in either sample. As a third example, the SHOOT MERISTEMLESS (STM) transcription factor is essential for SAM formation and maintenance  genes CESA4, CESA7, and CESA8 are associated with secondary wall synthesis , and CESA8  to be at least 125-fold enriched in the BR compared to the AR . Another well-established marker of xylem differentiation, XYLEM CYSTEINE PROTEINASE-2  were enriched 106-fold in the BR compared to the AR. Thus, expression of at least some well-known markers of early and late stem development were observed in patterns that matched expectations.To quantify the relative abundance of transcripts in the shoot apex (AR) as compared to the remainder of the stem (BR), the clean sequencing reads described in Section RNA Extraction and Sequencing were mapped to the flax reference genome  we used qRT-PCR to measure transcript abundance in independently grown replicates of the same tissues that were used for RNA-Seq. In order to select an appropriate reference gene for the qRT-PCR, GeNorm was used to determine the expression stability of nine commonly used reference genes among tissues assayed in our study  Discovery Grant 2014-03596 to MD; China Scholarship Council fellowship to NZ.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Reticulitermes labralis are investigated and compared. The results showed that the AR derived from the last instar nymphs by molting, and they were more similar to neotenic reproductives in morphology. In addition, the paired AR were not able to survive in the absence of workers. In R. labralis, the process of the last instar nymphs developing into AR and BN took an increase in JH level as a starting point. The JH level of the last instar nymphs molting into BN was approximately 1.5-fold higher than that of the AR. Additionally, The JHIII level of BN peaked on day 5, and that of AR peaked on day 10, which induced the onset of vitellogenesis in BN and AR, respectively. After molting, the vitellogenin gene expression levels of both BN and AR initially increased and then declined, and the expression levels in the BN were significantly higher than those in the AR. In addition, the oocytes of BN matured earlier than those of the AR, and the number of eggs laid by the BN was higher than the number laid by the AR. Our results demonstrate that, in R. labralis, the last instar nymphs can develop into AR, which are significantly different from BN in their development.Secondary reproductives develop primarily from nymphs. However, they have been rarely studied; in particular, the development of adultoid reproductives (AR) with floppy wings is still unclear. In this study, the change in juvenile hormone (JH) levels, vitellogenin gene expression, and oogenesis during the development of AR and brachypterous neotenic reproductives (BN) from the last instar nymphs of  Reticulitermes within Rhinotermitidae, as lower termites, is highly plastic, so that environmental stimuli can direct the developmental pathway  develop into workers or nymphs, which are distinguished by the absence or presence of wing pads, respectively. The primary reproductives develop via the last instar nymphs molting into winged sexuals  that fly off at the same time in great swarms, mate, and create new colonies. In the absence of the primary reproductives, some of the workers and nymphs develop into secondary reproductives to continue colony growth. The workers become apterous neotenics, and the nymphs become brachypterous neotenics (Macrotermes gilvus (Termitidae) developed from alate adults with torn wings and retained at the original nest as secondary reproductives. However, in Reticulitermes urbis and R. labralis, the AR with floppy wings develop from nymphs and have the secondary reproductive function  from the last instar nymphs are observed in field and laboratory colonies of R. labralis) were collected at Daxingshang Temple, Xi'an City, China, in April 2012 when the alate adults were flying off the rotten wood, and a large number of AR were found in the colonies. Three mature colonies of R. labralis were brought back to the laboratory, and the alate adults and AR were collected immediately. The wood was placed on the ground in the orchard of Northwest University under a humid, shaded area, and covered with moist sand soil.The experimental termites  appeared in the field colonies. To obtain AR and BN that developed from the last instar nymph under laboratory conditions, some of the last instar nymphs and workers were isolated from their natal colonies. One female last instar nymph and 10 workers formed a group (NW group) and were placed in 30 ml transparent bottles packed with sawdust at 25°C; 500 replications from three mature colonies were established.At 0 (time of molting), 5, 10, 15, and 20 d after the last instar nymphs in the NW groups molted to AR and BN and when eggs were found in the groups, the samples were collected, immersed immediately in liquid nitrogen, and stored at −80°C. Female last instar nymphs from the natal colonies and NW groups that were cultured for 5 d were also sampled and stored at −80°C for use in the JH and RNA extractions. For histological observations of the ovarian development of AR and BN, the samples were fixed using Bouin solution, stored at −4°C, and eventually stained with HE (hematoxylin and eosin).The degree of ovarian developments of the AR and BN was evaluated by counting the numbers of ovarioles. The oocyte development of the AR and BN was evaluated with HE staining. The fixed samples were dehydrated in an ascending ethanol series and embedded in paraffin. Longitudinal 7 μm sections were processed by a microtome and collected on polylysine-coated slides. The deparaffinized and rehydrated sections were stained with HE. The sections were observed using digital microscopes , and the number of vitellogenic oocytes was counted in each individual to evaluate the level of ovarian maturation.g for 5 min, the supernatant was collected and mixed with 10 µg of fenoxycarb  as an internal standard. The mixture was vortexed and allowed to stand at room temperature for 30 min before centrifugation at 5,600 × g for 15 min. The upper isooctane phase was transferred into a new glass vial, and the methanol phase was vortexed and centrifuged at 7,700 × g for 30 min and then combined with the isooctane phase. The resulting mixture was dried under a stream of nitrogen, and the dried pellets were dissolved in 20 µl of methanol.Previously reported extraction methods with modifications were applied . Briefly− 1. Quantification of JH III and fenoxycarb was performed by monitoring the [M + H]+ and [M+ Na]+ ions. A calibration curve for JH III  was plotted using the same internal standard concentration as fenoxycarb in each sample. The JH III level from each sample was then calculated after analysis of the chromatogram data using quantification software . Data are expressed as ng mg−1 fresh weight.The JH levels in the samples were analyzed using liquid chromatography-mass spectrometry (LC-MS) according to Total RNA was extracted from individual termites (removed intestines) and stored at −80°C using a FastPure RNA kit . Three different individuals were used for each category. After the DNase  treatment, the quality and quantity of the extracted RNA were determined by spectroscopic measurements at 230, 260, and 280 nm using a NanoVue spectrophotometer. cDNA was synthesized from 50 ng mRNA(DNase-treated) with an oligo-dT15 primer and Superscript II reverse transcriptase  and then incubated at room temperature for 10 min and 37°C for 30 min .R. flavipes . The relative quantification of the cDNA was performed using a SYBR Green I chemistry and LightCycler 480 System detection system .Quantitative polymerase chain reaction was performed as previously described  with betflavipes . PrimersFour groups of the female–male pairs of AR died after 1 d, and all the 50 pairs died within 2 wk. Tunnel digging behavior was not observed in the paired AR. The paired dealate adults started to dig tunnels in 3–7 d, and downward tunnels were clearly visible. After 2 wk, 44 pairs survived and only six pairs died. The paired alate adults laid eggs after 3 wk.R. labralis started molting after 1 wk, and 67.5% of last instar nymphs developed into AR and 32.5% developed into BN via a single molt in 9 wk. There were apparent morphological differences among the last instar nymph, AR, BN, and alate adult individuals. The last instar nymphs had a milky white body, long wing buds, and a lack of dark brown stripes on the head , oocyte growth stage (stage II), and oocyte vitellogenesis stage (stage III)  . In R. lstage II A and C. stage II D. On daystage II E. On daystage II F. On daystage II G. On daystage II H. Thus, n = 5); on day 15, the mean number increased to 3.3 ± 1.6 and 1.8 ± 0.3, respectively (n = 5), and on day 20, it reached 4.3 ± 0.6 and 3.3 ± 0.5, respectively (n = 5). The BN and AR began to lay eggs on day 22 and day 24, respectively; the mean number of BN and AR eggs was 2.7 ± 0.6 and 1.3 ± 0.5 (n = 5), respectively.On day 10, the mean number of vitellogenic oocytes of the BN and AR was 3 ± 0.7 and 0, respectively . However, M. gilvus (Termitidae) originated from alate adults with damaged wings and stayed in the natal colonies to become secondary reproductives; they were morphologically indistinguishable from the primary reproductives. The presence of wings might lead to the conclusion that the AR of R. labralis are primary reproductives , but their overall appearance is more similar to that of secondary reproductives (neotenic reproductives), and their wings are not functional. In R. labralis, it was observed that there were significant differences in level of exoskeleton sclerotization between the AR and alate adults. The insect exoskeleton is sclerotized. The exoskeleton as their major system for support, protection, and movement has profound effects on specific aspects of their method of growth, physiology, and muscle attachment  when the majority of the last instar nymphs become alate adults and disperse, and a small portion develop into AR remaining in the natal colony. Therefore, the early separation from the field colony may have accelerated last instar nymphs development towards sexual maturity and prevented their development into normal alate adults. The differentiation of AR in R. labralis expanded the developmental pathways of the last instar nymphs, making the mating system and life cycle of Reticulitermes more complex. We suggest that AR is a middle reproductive caste between the BN and the alate adults, which implicates evolutionary history of reproductive type in Reticulitermes.Not all species of termites produce AR. On studying  species . TherefoR. labralis, the process of the last instar nymphs developing into secondary reproductives takes an increase in JH level as a starting point, and compared with developing into AR, the last instar nymphs developing into brachypterous neotenics require higher JH levels, indicating that the level of JH influences the conversion direction of last instar nymphs in R. labralis. The last instar nymphs required dramatically increased JH levels before molting to reproductives. However, when the last instar nymphs of R. labralis were molting to AR and brachypterous neotenics, their JH levels were maintained at very low levels. The low JH titer during molting might result from the influence of the ecdysteroids because ecdysteroids may inhibit corpora allata (CA) activity by promoting the release of allatostatins or by causing variations in the sensitivity of the CA to the presence of the neuropeptides (R. flavipes, the workers that were destined to become pre-soldiers have 2.5-fold higher JH synthesis than the workers that would develop into neotenic reproductives (In peptides . The JH ductives .R. labralis, the JH levels of the brachypterous neotenics and AR reached their maximum values on days 5 and 10 after molting, respectively, whereas their vitellogenic oocytes occurred on days 10 and 15, respectively. Therefore, it is believed that the peak level of JH induces the initiation of oocyte vitellogenesis in R. labralis. In Orthoptera, the concentration of JH has been shown to be the critical factor that determines the onset and continuation of vitellogenesis (R. flavipes, CA with high rates of JH synthesis were from females with early vitellogenic oocytes, whereas CA with low rates of JH synthesis were from females with either pre-vitellogenic or mature oocytes (R. labralis are surrounded by a layer of follicle cells. It has been shown that JH regulates the uptake of vitellogenin by interacting with membrane receptors on the follicle cells in Locusta migratoria (R. labralis, the JH level of the reproductives with the developing oocytes was higher than that of the last instar nymphs from natal colonies; however, the JH level of the reproductives with oocytes at late vitellogenesis was lower than that of the last instar nymphs from natal colonies. This result is most likely due to the stimulation of JH synthesis by developing oocytes, and the inhibition of JH synthesis by almost mature oocytes.The appearance of the vitellogenic oocytes in the brachypterous neotenics was earlier than that in the AR, which was associated with the JH titer reaching its maximum value earlier in the brachypterous neotenics. In ogenesis . Previou oocytes , which i oocytes . 2014. Egratoria . JH stimgratoria . In addiR. labralis initially increased and then declined, the vitellogenin gene expression level of the brachypterous neotenics with lower JH levels was significantly higher than that of the AR with higher JH levels, indicating that the vitellogenin gene expression in brachypterous neotenics is very sensitive to JH induction. In addition, the JH levels of the brachypterous neotenics and AR peaked on days 5 and 10 after molting, respectively. Obviously, in the brachypterous neotenics, higher levels of the vitellogenin gene expression may be related to a faster peak in the JH level. Generally, in insects, the vitellogenin gene expression level is regulated by JH, which directly stimulates the transcription of vitellogenin genes in the fat body (Interestingly, although the vitellogenin gene expression levels of both brachypterous neotenics and AR of fat body . The vit


Timing variation in small group musical performance results from intentional, expressive, and unintentional, error components in individual player timing. These timing fluctuations produce variability in between-player note asynchrony and require timing adjustments to keep the ensemble together. The size of the adjustments relative to the asynchrony (correction gain) affects the amount and nature of asynchrony variability. We present new listening tests to estimate thresholds for perception of between-player asynchrony variability and to determine whether listeners use differences in the nature of the variability, as well as in its magnitude, to judge asynchrony. In two experiments, computer-simulated ensemble performances of a 48-note excerpt from Haydn Op. 74 No. 1 were generated. Between-player note asynchrony was systematically manipulated in terms of level of within-player timing variability (Experiment 1) and correction gain (Experiment 2). On each trial, participants listened to two samples, one (“target”) with more between-player asynchrony variability than the other (“test”), and reported which was “less together.” In both experiments, the test sample correction gain was fixed at the statistically optimal value of 0.25 and the within-player timing variability was minimal . In Experiment 1 the target correction gain was fixed at 0.25 and the timing variability was adjusted over trials by a staircase algorithm designed to converge on the level of asynchrony variability giving 75% correct identification. In Experiment 2 the timing variability in the target was set at half that in Experiment 1 and the correction gain was varied to converge on 75% correct identification. Our results show that the between-player asynchrony variability giving 75% correct identification in Experiment 2 was significantly lower than in Experiment 1. This finding indicates that people are sensitive to both the degree of variance and the micro-structure of the time-series of the asynchronies caused by differences in correction gain when judging lack of togetherness in quartet performance. Timing in ensemble musical performance is variable, both within and between players. A large part of this variability is intentional, arising from scored differences in note duration or players’ intentional departures from the score as part of musical interpretation, e.g., lengthening of notes when slowing the tempo at the end of a piece. However, some unintentional variation in timing may also be expected in the duration of notes  or as a result of tempo fluctuations. Although players may seek to reduce the level of unintentional variation by practice, it cannot be eliminated completely. In consequence, there will be small asynchronies between the note onsets of the various players. These asynchronies will vary from note to note, which may affect the listener’s perception of ensemble and this is the focus of the present paper.Recordings of various ensembles have shown that between-player asynchrony variability [measured in terms of SD] in professional ensembles playing various pieces from the repertoire  is typically in the 10s of milliseconds range, reducing with increasing tempo , 1998. IFigure 1). This correction is assumed to be a sum of proportional corrections:Although there is between-player asynchrony variability in quartet playing it appears to be stable over the course of a performance. It does not, for example, increase progressively throughout a piece of music . This imi,n and ti,n-1 are current and previous observed note onset event times for Player i, Ti,n-1 represents the timekeeper interval, αij refers to the correction gain applied by Player i for the asynchrony  with Player j, and 𝜀i,n is a random noise term identified with timing variability of intervals generated by an assumed internal timekeeper  lies between 0 and 0.5, the asynchrony on the next note tends to reduce and the asynchrony time series is stable. The size of the correction used by one player may differ for each of the other players. Thus more weight might be given to one player than another, for example because the timing of one is perceived as being more reliable than the other. Alternatively, the weight assigned to one player may be greater because that part is perceived as having greater musical significance  and it is considered important for non-leading parts to prioritize timing corrections with that leading part over corrections with each other.Figures 2A,B shows changes in the asynchrony variance of a virtual quartet performance of a 48-note excerpt of Haydn Op. 74 No. 1  simulated using Eq. (1) with two levels of within-player timing variability and across correction gains, which were set to be equal over all player pairs. It may be seen that the level of the between-player asynchrony variance can be equivalent for a quartet using less than optimal correction gains and a quartet whose members exhibit more within-player timing variability but whose correction gains are optimal .In the first-order linear correction model there are 12 proportional correction gains between all pairs of players. Theoretically, the variance of between-player asynchrony of a quartet is minimized if each player adopts a correction gain of 0.25 . SmallerFigure 3 we observed gain estimates which, on average, approximated the value of 0.25 . We varied the discrimination task difficulty up and down using a staircase paradigm to determine listeners’ thresholds for detecting differences in between-player asynchrony. In Experiment 1 gains were equal but there was a difference in within-player timing variability, and hence in the between-player asynchrony variability, which, over trials, was adjusted to give 75% correct identification. In Experiment 2 the timing variability was fixed at half the threshold level obtained in Experiment 1 and there was a difference in gains which, over trials, was adjusted to give 75% correct identification. We expected that the resulting difference in asynchrony variance obtained in Experiment 2 would be smaller than that observed in Experiment 1, despite the same accuracy of target identification, on the assumption that the listener is sensitive to the form, and not just the amount, of variance in note asynchrony.Figure 3 and then reported which sample (the “test” or the “target”) sounded less together (less well synchronized).Nine participants without specialized musical training , who provided informed consent according to ethics approved by University of Birmingham ethics committee, took part in the study. In two experiments , on each trial the participants listened to two samples of a virtual quartet performing the 48 note excerpt shown in 2 for all instruments. The target stimulus had non-zero asynchrony variance. This was adjusted on successive trials by an adaptive staircase algorithm using accelerated stochastic approximation . Feedback was given at the end of each trial as to whether the target stimulus had been correctly identified.We used the first-order linear correction model (Eq. 1) to generate the event times of the virtual quartet at a rate of 162 bpm while the variance of the asynchronies between the players was adjusted by changes in the within-player timing variability  was outside a tolerance region of the theoretical variance plus or minus 10-7 s2, then new stimuli were generated until one resulted with asynchrony variance within the tolerance region. In Experiment 1 the asynchrony variance threshold at which the listener correctly identified the target on 75% of trials corresponding to .The average tempo for the stimuli generated in Experiments 1 and 2 was 162.0 ± 3.3 bpm. For each participant, responses were divided into 10 bins by staircase levels, and the mean fraction of correct response was calculated per bin. The proportion of correct responses was then fitted with a logistic function and the 75% correct response level was determined as the detection threshold of between-player asynchrony variance (see t(8) = –20.848, p < 0.0005].Experiment 2 differed from Experiment 1 in that the correction gain was varied, which, in turn, affected the nature of variation in the asynchronies between players as revealed by the lag one autocorrelation. The largest of the lag one autocorrelations for the six pairs of between-player asynchronies was selected on each trial. The autocorrelation was reliably larger in Experiment 2  than Experiment 1 , [In a typical small group ensemble, the different instruments exhibit small variations in timing and this results in variable aysnchrony between the note onsets of the various player pairs. In this paper we present a method for determining the threshold at which listeners can discriminate between two virtual quartets, simulated using the first-order linear correction model. Using this method, Experiment 1 reveals that when the correction gains are optimal, the average threshold for discriminating between two musical excerpts differing only in between-player asynchrony variability is appreciably less (by a factor of 5 in terms of SD) than the published estimates of ensemble asynchrony variability referred to above. This suggests that listeners would be able to distinguish between typical quartets on the basis of differences in their asynchrony variability. Moreover, in Experiment 2, when the two musical excerpts differed in correction gain, they were discriminated at a point where the difference in asynchrony variability was at a level well below (by a factor of 2 in terms of SD) the threshold estimated in Experiment 1. This suggests that listeners are sensitive to the form of correction, probably because of the structure present in the variability of the asynchronies. For example, consider when the gain approaches zero, when discrimination against optimal gain becomes more reliable. The asynchronies exhibit persistent positive or negative departures from average (reliably larger lag one autocorrelation in Experiment 2 compared to Experiment 1), producing between player shifts in phase, and this may have been the cue used by participants.In Experiment 2 we examined correction gains in the range 0 to 0.25. Gains in this range produce damped convergence on zero asynchrony error. It would be interesting to know whether correction gains between 0.25 and 0.5, which result in alternation in the sign of the asynchrony as it reduces to zero with successive notes, would be more noticeable and lead to better discrimination of the target stimulus. It might be supposed that correction gains set by the individual player are subject to error. If deviations from the optimal value of 0.25 are less perceptible for the listener than when gains are greater than 0.25, players might adopt the strategy of setting their target gain less than 0.25. However, there are other possible reasons for setting the gain less than 0.25. For example, if there is appreciable variability in neuromuscular performance delays, the optimal value decreases with increasing motor variability .Our method of obtaining discriminability estimates involved a very low level of variability  in the test stimulus. This might be considered unrealistic for musical contexts where the discrimination task would typically involve identifying the more variable of two quartets, where both have appreciable asynchrony variability. The ability to discriminate target from test is likely to decline with increase in baseline variability by analogy with the increase in threshold for duration discrimination with duration . In futuIn summary we have presented new listening tests for the ability to discriminate variability of note onset asynchrony in string quartet performance. The results of the first experiment show that people discriminate differences in asynchrony variability that are appreciably less than the variability typical of professional quartets. The second experiment shows that discrimination is further improved if the performances differ in the correction gain employed to counteract timing variability and maintain ensemble.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand, and this calcium response is completely blocked by a selective β2-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and β2-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with β2-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by β2-AR antagonists, providing a new avenue for opioid therapy.The primary molecular target for clinically used opioids is the μ-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with β The μ-opioid receptor (MOR) is the main target for clinically used opioids, the most effective analgesic drugs for the treatment of moderate-to-severe pain. Long-term use of opioids, however, is compromised by adverse effects such as tolerance (reduced efficacy), physical dependence and opioid-induced hyperalgesia (OIH)1OPRM1 gene. MOR1 activation, which is pivotal for the analgesic effect of opioids, leads to reduced excitability of neurons by inhibiting adenylyl cyclase and voltage-gated Ca2+ channels and by activating of K+ channelsOPRM1 gene also codes for multiple alternatively spliced isoforms, including a new class of truncated isoforms, consisting of 6TM helices, lacking the first TM and the extracellular domain4The major isoform MOR1 is a seven-transmembrane (7TM) spanning G‐protein (Gαi/o)-coupled receptor (GPCR) encoded by the 2+ as well as increased nitric oxide production and release6At the cellular level, it has been shown that 6TM-MOR may play a particularly important role in altering MOR signaling52-AR for several reasons. First, β2-AR has been shown to chaperone other GPCRs; for example, physical and functional interactions between β2-AR and the CB1 cannabinoid receptor or the M71 olfactory receptor lead to trafficking of these GPCRs to the cell surface82-ARs in opioid tolerance, physical dependence and opioid-induced hyperalgesia in rodent models102-ARs are ubiquitously expressed enables co-expression with 6TM-MOR in the majority of mammalian cells including neurons1314Our search for the signaling partner for 6TM-MOR focused on the β2-AR and 6TM-MOR we combined computational analysis, biochemical and functional in vitro studies in BE(2)-C neuroblastoma transfected cells; ex vivo mice ganglion neurons and in vivo animal pain behavior tests. Our results provide evidence that the heterodimerization of 6TM-MOR with β2-AR underlies the molecular mechanisms for excitatory cellular signaling induced by opioids, which are blocked by β2-AR antagonists resulting in an enhancement of opioid analgesia and elimination of OIH.To study the putative interactions between β2-AR is able to chaperone 6TM-MOR to the cell surface. We expressed 6TM-MOR tagged with a FLAG epitope alone or in combination with each AR (untagged) subtypes  in HEK293 cells. We then screened the cells, using confocal microscopy, for 6TM-MOR localization. We observed that, when expressed alone, 6TM-MOR is primarily present in the intracellular compartment and co-localized with the endoplasmic reticulum (ER), and only a small fraction is found in the plasma membrane .Because 6TM-MOR is not localized to the cell membrane when over-expressed in heterologous cellular systems62-AR, we determined whether exposure to the non-selective β-AR agonist isoproterenol (ISO) causes internalization of the two receptors. Agonist stimulation of β2-ARs leads to rapid desensitization and trafficking of the receptors to lysosomes16172-AR-GFP co-transfected cells with 10 μM ISO for 30 min results in the internalization of both 6TM-MOR and β2-AR  in HEK293 cells stably expressing FLAG-tagged β2-AR. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibodies and the pulled-down proteins were then detected by Western blotting. The presence of β2-AR protein was confirmed in all three immunoprecipitates; strikingly, Myc immunostaining was observed almost exclusively in the sample obtained from Myc-6TM-MOR transfected cells (~35kDa), suggesting that 6TM-MOR, but not 7TM-MOR, is the primary partner for interacting with β2-AR -C cells and then quantified IBNtxA-dependent Ca2+ responses with real-time imaging following ligand application (2-AR showed a robust Ca2+ response that was significantly higher (25.2% of BE(2)-C cells, 10 μM IBNtxA) than in cells co-expressing 7TM-MOR and β2-AR (10.1% of BE(2)-C cells), or cells expressing only 6TM-MOR, 7TM-MOR, β2-AR, or EV -C cells, respectively; 2+ response -C cells responded to 1, 3 and 10 μM IBNtxA, respectively -C cells, respectively). Naloxone pre-treatment did not further inhibit IBNtxA response in cells that over-expressed either 7TM-MOR or 7TM-MOR/β2-AR, suggesting that, in the Ca2+ assay, IBNtxA acted specifically on 6TM but not on 7TM MOR. Pre-treatment with ICI 118,551 resulted in a significant inhibition of IBNtxA-induced Ca2+ responses in all treatment groups -Cresponse  were higectively . We next 118,551 . Pre-tret groups . These r2+ channels. Thus, neurons within these ganglia are important endogenous sites of action of the effects of opioids on nociceptive transmission2-AR in neurons isolated from naïve mice are low which brings challenges for functional assays in terms of obtaining reliable responses2+ responses in ex vivo neurons and determined if the evoked responses were dependent on β2-ARs  and trigeminal ganglia (TG) neurons encode nociceptive signals that give rise to the perception of pain, and are modulated by MOR activation via effects on voltage-gated Can β2-ARs . We harv2-AR heterodimer signaling, we performed in vivo animal studies to investigate the synergistic interaction between opioid (morphine and IBNtxA) and β2-AR  ligands using the mouse (5% plantar) formalin test. Because the activation of the 6TM-MOR/β2-AR heterodimer seemed to produce cellular excitatory signaling, we proposed that 6TM-MOR/β2-AR heterodimer signaling would contribute to a reduction in the analgesic efficacy of MOR agonists. We expected, therefore, that the co-administration of 6TM-MOR agonists and β2-AR antagonists would disrupt the excitatory dimer signaling, and produce analgesic synergy. To determine whether the interaction between IBNtxA and β2-AR antagonist ICI 118,551 is indeed synergistic, we conducted an isobolographic analysis. As expected, a dose-dependent analgesic response following the administration of either IBNtxA or ICI 118,551 alone was observed in both early . At this dose ICI 118,551 efficiently blocks βeriments . We obse partial . The veh partial . These r2-AR, and that this dimerization underlies a molecular mechanism for 6TM cellular signaling that mediates the excitatory and hyperalgesic effects of opioids. The co-expression and dimerization of these two receptors results in the translocation of the 6TM-MOR isoform from an intracellular compartment to the plasma membrane. The outcomes of our ex vivo studies suggest that this complex is expressed at low levels on a few sensory neurons. Stimulation of this complex by IBNtxA activates the signaling of 6TM-MOR/β2-AR heterodimers, which can be blocked by the selective β2-AR antagonist ICI 118,551. Finally, co-administration of 6TM-MOR and β2-AR ligands leads to substantial analgesic synergy and completely reverses OIH in animal models of pain.In the presented set of studies we provide evidence that the 6TM-MOR isoform heterodimerizes with β2-ARs in complex with olfactory receptorsB receptor subtypes1 and NTS2 receptors2-ARs is required for activation of wild-type M71 olfactory receptorsB receptor (GABABR1) is not effectively coupled to expected signaling pathways until co-expressed with GABABR2, which allows GABAB1R to escape from the ER311 creating a receptor complex responsive to neuromedin U30. Our data add another facet to current knowledge of GPCR heteromerization and demonstrate how receptor heteromerization may change the direction of opioid cellular signaling, from inhibitory to excitatory. Furthermore, because a specific isoform of the MOR receptor heterodimerizes with β2-AR unmasks a novel cellular signaling pathway, further investigation of the functional importance of 6TM isoforms in modifying GPCR signaling is warranted.The results have broad basic molecular and cellular implications. Although there has been a recent recognition that receptor heterodimerization adds another important dimension to the diversity of cellular responses controlled by GPCRs, the scope of these effects is just starting to emerge252+ channels on central presynaptic terminals of primary afferent nociceptors is thought to be one of the primary mechanisms mediating analgesia at the spinal level23Our findings may also have considerable clinical significance. Opioids remain the most commonly used drugs for treatment of moderate-to-severe pain. Opioid analgesia is largely facilitated by the inhibition of synaptic transmission, and MOR-mediated inhibition of voltage-gated Ca2-AR heterodimerization represents one of the molecular mechanisms underlying the excitatory cellular effects and produce enhanced nociception. Consistent with the cellular results, our animal data demonstrate synergistic analgesic interactions between 6TM-MORs agonist and β2-ARs antagonist. Furthermore, our findings also provide evidence that 6TM-MOR/β2-AR complex is signaling in vivo in response to repeated exposures to IBNtxA and contributes significantly to opioid mediated OIH as has been shown for morphine102-AR heterodimers. Whether the sensitization of primary afferent neurons by various analgesics produces an increase in the expression of 6TM-MOR/β2-AR dimers in primary sensory neurons is an interesting question that requires further investigation. However, it is clear that prolonged use of opioids up-regulates mRNA expression of the majority of MOR isoforms; for example in the brainstem, all 6TM variants were reported to be significantly increased (up to 100 folds), suggesting a greater effect upon the exon 11 promoter than upon the exon 1 promoter in this conditionsCurrent understanding of 6TM-MOR stimulation profile is favorable towards traditional inhibitory opioid mediated effects, and IBNtxA, like morphine, produces analgesia following acute administration by stimulating 6TM-MORs2-AR complex that functions to antagonize the analgesic properties mediated by 6TM- and 7TM-MORs. Consistent with this model, the reported improved adverse events profile of IBNtxA is related to its inhibitory effects associated with acute administration. The initial characterization of acutely administered IBNtxA concluded that respiratory depression and physical dependence are negligible and associated with limited inhibition of gastrointestinal transit was seen by IBNtxA3839The analgesic properties of IBNtxA in mouse models2-AR heterodimer can be efficiently blocked by a β2-AR antagonist and suggests that in the clinical setting, the simple co-administration of a β2-AR antagonist with opioid drugs may enhance the analgesic efficacy of opioids and diminish unwanted adverse effects, such as OIH.Thus, in accordance with our conceptual model, the hyperalgesic effects of an opioid agonist derived from 6TM-MOR/β2-AR activation. Circulating epinephrine, which is derived from the adrenal medulla, is the primary endogenous agonist for β2-ARs and mediates the effects of life relevant stressors on pain sensitivity41424445462-ARs plays a critical role in the production of opioid-induced tolerance and hyperalgesia, which are common clinical problems1011482-AR heterodimers play in these clinical states and in other clinical pain states that may result in the expression of 6TM-MOR/β2-AR .Finally, our findings have profound implications on understanding the molecular processes that contribute to stress and opioid induced hyperalgesia, which have been linked to βIn summary, our results contribute to the emerging effort to identify new therapeutic targets that selectively modulate inhibitory opioid receptor signaling while reducing the adverse effects associated with opiate use.2-AR was a gift from Dr. R. Lefkowitz 2/95% air in F12 media (Life Technologies) with 5% FBS.HEK293T and human neuroblastoma BE(2)-C cells  were maintained and transfected with Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s instructions as described previouslyOPRM1 6TM-MOR mRNAs: OPRM1K1, OPRM1K2, OPRM1-JL, OPRM1G1, but not MOR-3 and OPRM1-W  or OPRM1G2 . N-terminus FLAG-tagged (DYKDDDDK) 6TM WT and series of 6TM mutants . β2-AR mutants 1) I201A; 2) I201A,V216A, I291A, V295A; 3) O205A, S220A, Q224A, I298A, L297A; 4) V117I; 5) F298Y; 6) F290T; 7) V117I, F298Y, F290T were generated by site-directed mutagenesis  and cloned into the pOPRSVI/MCS vector. All constructs were verified by double-stranded DNA sequencing.Human pIRES-EGFP-7TM and -6TM-MOR (no tag) expression constructs, and pIRES-EGFP-Myc-7TM and -6TM constructs were described previously mutants  1) I234A2-AR as described above. 48 h after transfection, cells were fixed in -20°C methanol for 10 min, permeabilized with 0.25% Triton X-100 and blocked with 5% BSA for 40 min followed by immunostaining . Primary anti-FLAG (DYKDDDDK) antibody (Sigma-Aldrich) was used at a 1:1,000 dilution, anti-Calnexin (Abcam) and anti-Na/K ATPase α1 (Santa Cruz Biotechnology) antibodies were diluted 1:300, and anti-β2-AR 1:500 (Thermo Fisher Scientific). After washing with TBS, coverslips were incubated with appropriate secondary antibodies conjugated to Alexa Fluor 488 or 594 (Life Technologies) together with Hoechst 33342 (Sigma-Aldrich) for 1 h at room temperature. Samples were imaged using a Zeiss LSM 710 confocal microscope and images were collected and analyzed with ZEN Black software (Carl Zeiss).HEK293T cells were plated onto glass coverslips and transfected with plasmids encoding FLAG-tagged 6TM-MOR alone or with pOPRSVI/MCS-β2-ARs2-AR  and anti-Myc (EQKLISEEDL) antibodies .HEK293 cells stably expressing FLAG-tagged βB (~300 K). With the exception of atoms belonging to the i3 loop, all atomic coordinates from the crystal structure were harmonically constrained. We generated a total number of 1,000 loop conformations and, performed a cluster analysis based on the root mean squared distance (RMSD) of backbone-Cα atoms (applied cutoff of 2.5 Å). Finally, we retrieved the centroid of the most populated cluster as the representative conformation of the i3 loop in the 6TM-MOR structural model. We then assessed the quality of our generated model using Gaiahttp://troll.med.unc.edu/chiron/login.php), which compares their intrinsic structural properties to high-resolution crystal structures. No critical issues were found in our 6TM-MOR model.We generated the structural models of 6TM-MOR by applying minor changes to the crystallographic coordinates of the MOR chimera (PDB ID: 4DKL)22202-AR at 2.8 Å resolution (PDB ID: 3NY80)522-AR heterodimer by performing protein-protein docking calculations using the online server ZDOCKet al., we restricted the docking exploration to protein surfaces corresponding to the transmembrane helices 5 and 6 in both 6TM-MOR and β2-AR. We retrieved the docking top-ranked solution of the 6TM-MOR/β2-AR heterodimer, and then optimized its structure using DMD202-AR heterodimer.Starting from the X-ray structure of β2-AR (6TM/β2-AR), 7TM/pIRES-EGFP-EV (7TM/EV), pIRES-EGFP-7TM/pOPRSVI/MCS-β2-AR (7TM/β2-AR) or pOPRSVI/MCS-β2-AR/pIRES-EGFP-EV β2-AR/EV) expression plasmids. BE(2)-C cells or dissociated neurons were seeded onto glass coverslips, loaded with 1μM of the cell permeable calcium sensitive dye, Fura 2AM (Life Technologies) for 30 minutes and washed with PBS or HBSS before use. Coverslips containing cells were placed in a chamber with constant infusion of PBS or HBSS at room temperature as indicated below. Perfusion conditions for BE(2)-C cells were: 1 min PBS; 59 min 1, 3 or 10μM IBNtxA  or 1 min PBS-3 min 10 μM naloxone or 1μM ICI 118,551 (Sigma Aldrich); -56 min 10 μM naloxone/1μM IBNtxA or 1μM ICI 118,551/1μM IBNtxA as indicated. Perfusion conditions for mouse trigeminal and dorsal root ganglia neurons were: 1 min HBSS, 18 min 50μM IBNtxA or 1 min HBSS-3 min 0.3μM Betaxolol HCl (Tocris Bioscience), or 0.3μM ICI 118,551 or 0.1μM SR-58611A (Tocris Bioscience). For neuronal imaging, 75mM KCl was used as a positive control at the end of the recording to ensure the responsiveness of the recorded cells. Fluorescence was detected by a Nikon Eclipse TI microscope at 340 and 380 excitation wavelengths and analyzed with the TI Element Software (Nikon). Cells were considered as responsive to a drug infusion if the 340/380 ratio was ≥0.2 from baseline. Mean values were compared using one-ANOVA, Sidak’s multiple comparison test or Student t test.BE(2)-C cells were transiently transfected for 24 hours with pIRES-EGFP-EV alone or cotransfected with pIRES-EGFP-6TM-MOR/pIRES-EGFP-EV (6TM/EV), pIRES-EGFP-6TM-MOR/pOPRSVI/MCS-βNaïve young adult (6–14 week old) outbred CD-1 (ICR: Crl) mice (Charles River) were used in drug interaction (isobolographic) studies. Approximately equal numbers of males and females were used in these behavioral experiments. Mice were housed in groups of 2 to 4, with same-sex littermates in standard polycarbonate cages. Subjects were housed in a temperature (20 °C ± 1 °C) and light-controlled  environment with tap water and food (Harlan Teklad 8604) available ad libitum. All procedures were consistent with appropriate national guidelines and approved by the McGill Downtown Animal Care and Use Committee.For OIH studies C57bl/6J male mice  were used. Mice were 7 weeks old upon the arrival and were allowed to recover for 7 days before the experiments. The mice were housed in plastic cages (n = 1–3/cage) with controlled ambient temperature of 23 ( ± 2)°C. Light-dark cycle was fixed to 12 h and all experiments were conducted during the light phase of the day, between 13:00 and 18:00 h. Water and standard laboratory chow were available ad libitum. Animals were not pre-selected for nociceptive experiments. After 5 days of habituation to the testing environment and equipment, sensitivity to mechanical stimuli was assessed. Based on baseline mechanical sensitivity, mice were allocated to the balanced treatment groups. Experiments were conducted in accordance with local and European regulation, guidelines of the International Association for the Study of PainIBNtxA , SR 59230A , Betaxolol HCl  and ICI 118,551  were dissolved in physiological saline and administered intraperitoneally in a 10 ml/kg injection volume in the formalin model. Experimenters were blinded to drug and dose during testing. For OIH studies morphine hydrochloride was obtained from the University Pharmacy ; morphine, IBNtxA and ICI 118,551 in this study were dissolved in physiological saline (0.9%) and administered in volume of 100 μl subcutaneously.1-, β2-, and β3-AR antagonists alone, and for IBNtxA. Half-maximal analgesic doses (AD50s) and associated 95% confidence intervals (CIs) and potency ratios were calculated using the method of Tallarida and Murray1-, β2-, and β3-AR antagonists using isobolographic analysis. First, AD50 values we determined for each drug administered alone. Equipotent doses of IBNtxA and each β-adrenergic antagonist were then calculated based on the AD50 dose-ratio, yielding a ratio of 1:13 between both IBNtxA and betaxolol and IBNtxA and ICI 118, 551, and a 1:10 ratio between IBNtxA and SR59230A. IBNtxA/β antagonist cocktails were then mixed at these ratios and administered to mice as described above. To determine whether the interaction between IBNtxA and the β1-, β2-, and β3-AR antagonists is synergistic, we used the AD50s of each drug separately to determine a theoretical additive value for the combination, and compared this value to the experimental AD50 of the cocktail we recorded. If the observed AD50 was significantly lower (p < 0.05) than the theoretical additive value, and then the interaction was confirmed to be synergistic. Isobolographic analyses were performed using the method of Tallarida and MurrayMice were habituated in 8 individual Plexiglas cylinders placed on top of a glass surface suspended over high-resolution video cameras for at least 30 min prior to experimentation. Mice were then removed from their cylinder, injected with IBNtxA, betaxolol, ICI 118,551 or SR59230A alone, or in cocktails of equipotent doses (interpolated from the single drug dose-response curves) and placed back in the cylinder. Fifteen min later, all subjects were injected subcutaneously into the left plantar hind paw with 5% formalin (20 μL volume). Mice were subsequently videotaped for 60 min, and videotape observations were later sampled for 10 sec at 1-min intervals, with an observer blind to experimental condition recording the presence or absence of left hindpaw licking/biting in that 10-sec period. The early phase of the formalin test was defined as 0 to 10 min after injection, and the late phase as 10 to 6 min post-injection. Data are presented as the percentage of samples licking in either the early and late phase of the formalin test. In statistical analysis, a criterion α = 0.05 was adopted. Dose-response curves were created for each βet al. 2006; 2007, as they have been reported to produce profound opioid induced hyperalgesia. Animals were administered morphine (20mg/kg) or IBNtxA (2mg/kg), or vehicle twice a day (8 am and 16 pm) for three consecutive days. For the fourth and fifth days the dose was increased to 40 mg/kg (morphine) and 4 mg/kg (IBNtxA). IBNtxA dose was selected based on the assessments of its acute analgetic potency in this study . Δ cumulative response rate was used as an outcome measure in statistical analysis and group comparisons.To measure mechanical allodynia, the mice were placed on a metal mesh and covered with individual transparent plastic domes enabling the observation of the nociceptive behavior and stimulation of the paw with a calibrated von Frey monofilament . Monofilament was slowly brought into contact with skin, perpendicularly to the surface. After skin contact, force just sufficient to bend the filament was applied for 3 s to standardize the time-course of stimulation. Determined foot withdrawal and brisk shaking or licking of the paw not associated with locomotor movement were accepted as a nociceptive response. Monofilaments ranging 0.008–4 g were applied in ascending order. Each filament was applied five times with 2-second interval between stimulations, and 5 min interval between filaments. The response rate for each monofilament was calculated: if the animal did not respond to any of five stimulations, the response rate for the given filament was 0%. Consequently, if all of the five stimulations induced nociceptive responses, response rate was 100%. Further, response rates of all tested filaments were counted together yielding the cumulative response rate, which was calculated for each animal. Further, the cumulative response rate was calculated for each animal as Thermal and cold sensitivity were assessed using hot/cold plate test . In the hot plate test, mice were placed on the heated metal surface and covered with transparent plexiglass dome. Licking or brisk shaking of the hind paw, or jumping, was considered as a sign of thermal nociception and latency to the first reaction was measured. To avoid tissue damage, the cut-off time was set to 60 s. Basal thermal nociception in the hot plate was assessed in morphine or IBNtxA treated animals on the fifth day of the experiment. The temperature of the hot plate was set to 50 °C (±0.2 °C), based on the results of pilot experiments. Cold nociception was assessed in similar manner at temperature of 3 °C or −5 °C for morphine or IBNtxA respectively, 30 min after the hot plate test. Temperatures were selected based on pilot experiments.t test and two-way ANOVA combined with Tukey’s multiple comparison test, depending on the number of groups. GraphPad Prism 6.0 was used for all data analyses.The level of significance was set at P value of 0.05. Prior to main analysis, the data gathered from nociceptive assays of OIH-experiments was checked and confirmed to meet the assumptions of parametric analysis. Normality of distribution was tested with Shapiro-Wilk normality test and the equality of variances with F-test. In assays of thermal and mechanical nociception, there were no differences between the results of vehicle groups of IBNtxA and morphine experiments. This allowed us to pool the results of vehicle groups in order to increase statistical power. In the comparisons between the treatment groups, mean values were compared using Student two-tailed How to cite this article: Samoshkin, A. et al. Structural and functional interactions between six-transmembrane µ-opioid receptors and β2-adrenoreceptors modulate opioid signaling. Sci. Rep.5, 18198; doi: 10.1038/srep18198 (2015).


AR) gene, the disease-causing AR disrupts axonal transport by acting in both a cell-autonomous fashion in the motoneurons themselves, and in a non-cell-autonomous fashion in muscle. The non-cell-autonomous mechanism is suggested by data from a unique “myogenic” transgenic (TG) mouse model in which an AR transgene expressed exclusively in skeletal muscle fibers triggers an androgen-dependent SBMA phenotype, including defects in retrograde transport. However, motoneurons in this TG model retain the endogenous AR gene, leaving open the possibility that impairments in transport in this model also depend on ARs in the motoneurons themselves. To test whether non-cell-autonomous mechanisms alone can perturb retrograde transport, we generated male TG mice in which the endogenous AR allele has the testicular feminization mutation (Tfm) and, consequently, is nonfunctional. Males carrying the Tfm allele alone show no deficits in motor function or axonal transport, with or without testosterone treatment. However, when Tfm males carrying the myogenic transgene (Tfm/TG) are treated with testosterone, they develop impaired motor function and defects in retrograde transport, having fewer retrogradely labeled motoneurons and deficits in endosomal flux based on time-lapse video microscopy of living axons. These findings demonstrate that non-cell-autonomous disease mechanisms originating in muscle are sufficient to induce defects in retrograde transport in motoneurons.Defects in axonal transport are seen in motoneuronal diseases, but how that impairment comes about is not well understood. In spinal bulbar muscular atrophy (SBMA), a disorder linked to a CAG/polyglutamine repeat expansion in the androgen receptor ( Thus, one trait of diseased motoneurons, impaired axonal transport, can be conferred by disease processes originating in muscle.Our findings suggest that therapies targeting skeletal muscle could potentially rescue motoneurons from axonal transport dysfunction in neuromuscular disease. Axonal transport is critical for proper motoneuronal functioning and is often impaired in neurodegenerative disease, including spinal bulbar muscular atrophy, an androgen-dependent neuromuscular disease linked to a polyglutamine expansion in the androgen receptor. In this study, we show that AR activated by androgens exclusively in skeletal muscle is sufficient to trigger defects in retrograde transport in the motoneurons. Specifically, diseased mice show impaired retrograde labeling of motoneurons AR) gene  gene . SBMA ocAR) gene . With onAR) gene , studiesAR) gene . How AR AR) gene  and/or iAR) gene . UnderstThat non-cell-autonomous mechanisms can instigate motoneuronal dysfunction in neuromuscular disease has been the subject of several recent reviews . BecauseAR gene, it leaves open the possibility that the effect of androgen on motoneuronal retrograde transport may have also depended on ARs acting in other cell types, including the motoneurons themselves. To eliminate this possibility, we generated mice in which the only source of functional ARs was from the transgene that is expressed only in muscle. We did this by passing the transgene into males carrying an endogenous allele for AR that is dysfunctional, the testicular feminization mutation (Tfm), providing a null background for functional ARs. Genetic males carrying the Tfm allele are insensitive to androgens, exhibiting a female phenotype, but have normal motor function , have functional ARs only in a single cell type—skeletal muscle fibers. This strategy allowed us to ask whether a disease-causing AR allele expressed solely in muscle fibers is sufficient to impair axonal transport in motoneurons via a non-cell-autonomous mechanism.Reports indicate that retrograde transport is impaired in an androgen-dependent fashion, paralleling the loss of motor function, in a myogenic model of SBMA . This unfunction . Thus, mUsing cholera toxin (CT) to label retrogradely transporting endosomes, we found fewer labeled motoneurons in the lumbar spinal cord of motor-impaired Tfm/TG males than in the lumbar spinal cord of Tfm-only control males with intact motor function, a deficit in Tfm/TG males that is comparable to prior results in myogenic TG males on a background of WT AR . Analysiin vitro fertilization. Sperm was harvested for in vitro fertilization from a TG male that overexpresses WT ARs specifically in muscle cells  and, thus, were exposed to the same prenatal flutamide treatment as Tfm/TG males. Because Tfm male mice develop a deficit in androgen production postnatally . Such T implants result in serum T levels comparable to that of adult gonadally intact males . After 5m phosphate buffer (pH 7.4) containing 0.8% paraformaldehyde and 1.25% glutaraldehyde. Spinal cords were dissected out and placed in the same fixative for 5 h at 4°C, after which they were transferred to a 10% phosphate-buffered sucrose solution and held overnight at 4°C. The following day, spinal cords were transversely sectioned on a freezing, sliding microtome at 40 μm. Sections were reacted with tetramethylbenzidine (TMB) for histological visualization of HRP  into the anterior tibialis (AT) muscles of isoflurane anesthetized mice. Once mice recovered from anesthesia, they were returned to their home cage. After 12 h to allow CT-HRP to retrogradely transport to motoneuronal cell bodies, mice were reanesthetized with an intraperitoneal injection of sodium pentobarbital. Once deeply anesthetized, mice were perfused with 0.9% saline followed by cold 0.1 n of HRP . SectionEndosomal trafficking in sciatic nerve axons of diseased (T-treated Tfm/TG) and healthy (T-treated Tfm) male mice was assessed by directly monitoring the retrograde transport of endosomes in living axons. CT B conjugated to Alexa Fluor 488  was injected into the AT and gastrocnemius muscles of isoflurane-anesthetized mice to label endosomes. Retrograde movement of endosomes in living axons of explanted sciatic nerves was monitored 4 h after injection. During the 4 h delay, mice were awake and mobile.m: 135 NaCl, 5 KCl, 1 MgCl2, 1.5 NaHCO3, 1 Na2PO4, 2 CaCl2, and 1.1 glucose, pH 7.2). The right sciatic nerve was harvested first and recordings were made from it, while the left nerve remained in situ in the anesthetized mouse. There is no evidence that transport in left and right nerves systematically differs or that transport declines over time (data not shown), which is consistent with previous findings (Mice were re-anesthetized with isoflurane to harvest the sciatic nerve. Explants (∼10 mm in length) were placed on coverglass, glued in place , and covered with 37°C oxygenated bicarbonate Ringer’s solution  equipped with a 60× PlanApo oil-immersion objective , illuminated with the 488 nm line of a 150 mW argon laser (Melles Griot), and recorded with a Photometrics CoolSNAP HQ2 camera using NIS Elements software. Temperature of the sciatic nerve explant was maintained at 37°C by a ring incubator. Less than 10 min after nerve harvest, recordings began, with two time-lapse movies taken from the same explant within 30 min of harvest. Each movie was 7 min in length, comprising 211 images, with images captured every 2 s at a 1 s exposure. Laser power and aperture settings were identical for all captured images. Scanning for trafficking started at the center of the sciatic nerve segment and proceeded toward the distal end of the nerve to reduce variability. Because of concerns about photobleaching, different axons in the same explants were used for the two movies, and the second movie was taken from a region distal to the first. To minimize experimenter bias, video capture began when the first moving endosome could be kept in focus.z-projected using the SUM option, which resulted in 32 bit “raw” kymographs. The raw kymographs were converted to 16 bits, and saved as tiff files. To facilitate the visibility of transport events, raw kymographs were opened in Adobe Photoshop (version 7.0 or CS2), color inverted, and resampled to increase size by 500%, thus producing the kymographs used for analysis, using ImageJ. We measured flux, net, and instantaneous velocity, run length, and frequency of trafficking perturbations  for retrogradely transporting endosomes.Time-lapse movies were converted into kymographs, single two-dimensional images showing the movement of endosomes  along the length of an axon as a function of time . To makeEndosomal flux is the number of retrogradely transporting endosomes in an axon per unit of time and was determined by drawing a line through the center of the kymograph parallel to the time axis and counting the number of endosomal traces that cross that line. The total number of endosomes per kymograph was then divided by 7 min  to estimate endosomal flux as the number of endosomes per minute. Such estimates were averaged across individual axons within a single animal to obtain a single estimate of flux per mouse. Net velocity of retrogradely transported endosomes is the net distance traveled by each endosome in the distal to proximal direction over time and included stalls, reversals in direction, and/or changes in velocity that might have occurred. Measures of net velocity were obtained from kymographs by drawing a box around each endosomal trace such that the far ends of the trace pass through opposite corners of the box. The height and width of the box (in pixels) corresponding to length and time are used to calculate velocity (in microns per second) based on the following formula: [distance (pixels) × 0.10526 (microns per pixel)/time (pixels) × seconds per frame]. Net velocities of individual endosomal traces in a single kymograph were then averaged to provide the mean estimate of the net velocity per axon. These estimates were then averaged across individual axons sampled within a single animal to obtain a single estimate of net velocity per mouse.Instantaneous velocity was measured by drawing a 1-pixel-wide line parallel to the time axis, bisecting the kymograph with respect to distance. For each endosomal trace crossing the center line, a 20 pixel box was drawn around it, centering it with respect to the bisecting line, with width and height of the box representing distance and time, respectively. The width of the box (20 pixels) accounted for 6.6% (1.67 mm) of the total distance traveled, and any continuous velocity within it was considered instantaneous. The time it took to travel this short distance was calculated based on the height of the box, which was adjusted so that its corners intersected the most distal and proximal aspects of the trace. On rare occasions, perturbations in endosomal velocity, including changes in speed, stalls, and/or reversals, appeared within the box. In such cases, the box was moved immediately distal to the trafficking perturbation or until the box included a part of the endosomal trace that contained no trafficking perturbations. Instantaneous velocity (in microns per second) was calculated based on the same formula used for net velocity. Measures of instantaneous velocity for each endosome were averaged within a single kymograph, and such estimates from two to four axons of the same mouse were averaged to obtain a single estimate of instantaneous velocity per mouse.N is the number of animals/group. Analyses of kymographs were performed by an experimenter blind to animal identification and genotype. Refer to the statistical table  and continues to drop progressively during the next 4 d a, exhibiting the same time course of demise as seen in T-treated myogenic TG females b. Interestingly, the number of labeled motoneurons for Tfm control males is comparable with that previously reported for gonadectomized WT males, who lack testicular androgens (Muscles of Tfm/TG mice and Tfm controls were injected with CT-HRP at the end of treatment (5 d) to assess the efficiency of transport based on the number of retrogradely labeled motoneurons. We find that motor-impaired Tfm/TG males have a significant deficit in the number of retrogradely labeled motoneurons compared with Tfm controls c, but not net velocity (p = 0.411)d, is significantly impaired in sciatic nerve axons e, an outcome that was not apparent in myogenic TG males (p = 0.0387)f, which is caused by more endosomes in diseased Tfm/TG males showing changes in velocity (p = 0.0435)g and reversals (p = 0.0314)h compared with endosomes in the axons of healthy Tfm males. Surprisingly, although more endosomes tend to stall in diseased axons than in healthy axons, this difference was not significant (p = 0.0955)i. Likewise, while the mean run length of endosomes in Tfm/TG is shorter, it too was not significantly decreased compared with that of healthy Tfm males (p = 0.2784)j. Together, these data suggest that endogenous WT ARs may have protected against some of the effects of a toxic muscle AR on transport, and converge on the conclusion that AR acting only in muscle fibers is sufficient to impair retrograde transport/trafficking of endosomes in motoneurons.We also measured instantaneous velocity and trafficking perturbations and unexpectedly detected a deficit in instantaneous velocity , thus eliminating all functional AR from the endogenous gene. Consequently, such Tfm/TG males had functional AR only in skeletal muscle fibers due to the expression of the AR transgene. We found that diseased Tfm/TG males showed comparable deficits in the number of retrogradely labeled motoneurons and in endosomal flux as previously reported for diseased TG males on a WT AR background , glial cell line-derived factor, insulin-like growth factor 1, neurotrophin (NT)-4, and vascular endothelial growth factor (VEGF) . MoreoveLoss of such trophic support from target musculature could lead to defective transport in at least two ways. First, lack of trophic factor signaling might mean a loss of survival signaling, leaving the cell deprived of needed resources for proper maintenance of axonal transport . AlternaWhile considerable attention has recently been redirected to muscle, recent work also supports the idea that mutant AR acting in motoneurons contributes to SBMA pathogenesis . Given t