Atrial fibrillation (AF) is the most common arrhythmias among old people. It causes serious long-term health problems affecting the quality of life. It has been suggested that the autonomic nervous system is involved in the onset and maintenance of AF in human. However, investigation of its pathogenesis and potential treatment has been hampered by the lack of suitable AF models in experimental animals.Our aim was to establish a long-lasting AF model in mice. We also investigated the role of adrenergic receptor (AR) subtypes, which may be involved in the onset and duration of AF.2+ leak, a major trigger of AF, and consequent spontaneous SR Ca2+ release (SCR) in atrial myocytes. Consistent with the results of our in-vivo experiments, both metoprolol and prazosin significantly inhibited the NE-induced SR Ca2+ leak and SCR. These findings suggest that both β1-AR　and α1-AR may play important roles in the development of AF.Trans-esophageal atrial burst pacing in mice could induce AF, as previously shown, but with only a short duration (29.0±8.1 sec). We found that adrenergic activation by intraperitoneal norepinephrine (NE) injection strikingly increased the AF duration. It increased the duration to more than 10 minutes, i.e., by more than 20-fold . In this model, a prior injection of a specific β1-AR blocker metoprolol and an α1-AR blocker prazosin both significantly attenuated NE-induced elongation of AF. To further explore the mechanisms underlying these receptors’ effects on AF, we assessed the SR CaWe have established a long-lasting AF model in mice induced by adrenergic activation, which will be valuable in future AF study using experimental animals, such as transgenic mice. We also revealed the important role of β1- and α1-AR-mediated signaling in the development of AF through in-vivo and in-vitro experiments. Atrial fibrillation (AF) is the most common arrhythmias, especially among elderly people, and causes harmful effects to the patients ,2. The l2+ leak, which is known to be a major trigger for AF, and the consequent spontaneous SR Ca2+ release (SCR) in mouse atrial myocytes. Our results have indicated that not only β1-AR but also α1-AR-mediated signaling are involved in the NE-induced SR Ca2+ leak and SCR as well as maintenance of AF.Numerous studies have demonstrated that activity of the autonomic nervous system is closely involved in the onset and maintenance of AF . HoweverMale C57BL/6 mice aged 11–12 weeks were purchased from Japan SLC . Standard food and water were provided ad libitum to mice. Twelve- to 14-week-old male mice were used for the experiments performed in this study. All animal experiments were approved by the Animal Care and Use Committee of Yokohama City University School of Medicine.Simple and minimally invasive AF models have been established in small animals such as rats and mice. We induced AF using rapid transesophageal atrial pacing according to previously reported methods with some modifications . BrieflyAll the reagents used in this study were purchased from Sigma Aldrich  unless described otherwise. For sympathetic activation, norepinephrine (NE) bitartrate dissolved in natural saline  was intraperitoneally injected 10 minutes before the induction of AF. For the selective blockade of adrenergic receptor (AR), mice were intraperitoneally injected with either 2 mg/kg of metoprolol (a β1-AR-selective antagonist), 1 mg/kg of prazosin (an α1-AR selective antagonist), or natural saline 45 minutes before NE administration.2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 12 mM NaHCO3, 20 mM D-glucose, 10 mM HEPES, 30 mM taurine, 2 mM creatinine, 2 mM carnitine and 5 mM butanedione monoxime (pH 7.4). The atria were cut into several small pieces and incubated in JMEM containing 0.02 mg/mL Liberase TH  with occasional agitation for 60 minutes followed by gentle trituration for 5 minutes at 37°C. The same volume of JMEM with 1% (w/v) BSA was added and the suspension was filtered through a 100 μm mesh . Cells were precipitated by centrifugation for 2 minutes at 40 x g and the pellet was gently resuspended in JMEM with 1% (w/v) BSA. Ca2+ reproduction was gradually performed to a concentration of 1.25 mM. Myocytes were precipitated again and resuspended in attaching media consisting of Medium 199 (Life Technologies) with 4% (v/v) FBS and 1% (v/v) penicillin/streptomycin (Wako). The cells were then plated onto laminin-coated glass cover-slips and incubated at 37°C in humidified air with 5% CO2 for 1 hour. Finally, attaching media was exchanged for maintaining media (Medium 199 containing 1% BSA and 1% penicillin/streptomycin). The prepared myocytes were bathed in maintaining media at 37°C before Ca2+ transient was measured.Atrial myocytes were prepared from adult mice as previously described with some modifications ,12. Brie2+ transient was performed according to previously reported protocols with several modifications [2, 10 mM glucose and 10 mM HEPES, pH 7.4 adjusted with NaOH) containing 1.8 mM Ca2+ for 15 minutes. Cells were washed twice with normal Tyrode solution and transferred to a chamber equipped with platinum electrodes. The chamber was placed on a Ti2000 confocal microscope system  in a dark room. To detect SCRs, the external solution was rapidly switched from normal Tyrode to 0Na+/0Ca2+ Tyrode . Myocytes were bathed for 30 seconds in 0Na+/0Ca2+ Tyrode solution, and SCRs were counted during this period. After SCR measurement, diastolic Ca2+ leak from the SR was estimated by a quick treatment with tetracaine. The ryanodine receptor (RYR) was rapidly and reversibly blocked by 1 mM tetracaine, causing Ca2+ uptake from the cytosol into the SR. The tetracaine-dependent shift of Ca2+ from the cytosol to the SR was considered to be proportional to the SR Ca2+ leak [2+ content.The measurement of Caications ,13. All 2+ transient. The protocol described above for the measurement of SCR and Ca2+ leak was repeated using normal Tyrode or 0Na+/0Ca2+ Tyrode solution supplemented with AR antagonist (1 μM) and NE (1 μM).For drug-treated samples, myocytes were pre-incubated with 1 μM of AR antagonist (prazosin or metoprolol) and 1 μM of NE for 5 minutes prior to the measurement of Cat test (two-tailed) or one-way ANOVA followed by the Tukey-Kramer post-hoc study for multiple comparisons. P value <0.05 was considered to indicate statistical significance in this study.All values were represented as mean±SEM. All statistical analyses were performed by Student’s AF was inducible by trans-esophageal pacing, as previously reported . AF was With the above-mentioned method, AF was reliably induced, but with a short duration, as pointed out in previous studies ,17. The Because it has been suggested, in human, that autonomic imbalance may trigger the onset and duration of AF, we investigated the effect of adrenergic activation by intra-peritoneal NE injection. We found that NE administration strikingly and stably elongated the duration of AF in a dose-dependent manner . The AF To determine which types of AR-mediated signaling play important roles in the NE-induced elongation of AF, we examined the effects of prazosin and metoprolol on the duration of AF in our model. The doses of metoprolol and prazosin were determined based on previous reports ,19. The 2+ leak and SCR in isolated atrial myocytes. The magnitude of the diastolic Ca2+ leak from SR was expressed as a value relative to the caffeine-releasable SR Ca2+ content [2+ leak  and the rate of SCR   and SCR  in a dose-dependent manner. In addition, prazosin treatment also significantly attenuated the SR Ca2+ leak  and SCR   . Consist P<0.01) , treatmeP<0.001) . These re.g. intravascular catheterization [In this study, we established an adrenergic activation-induced long-lasting AF model in mice. NE injection significantly elongated transesophageal burst pacing-induced AF from around 30 seconds  to 10 mirization . Thus, wUsing the model, we next investigated which AR subtype was more prominently involved in AF maintenance. The sympathetic and parasympathetic nervous systems play pivotal roles in the development of AF . Consist2+ leak and the consequent spontaneous SR Ca2+ release in cultured atrial cardiomyocytes. Catecholamine-induced phosphorylation of RYR by protein kinase A or Ca2+/calmodulin-dependent protein kinase II (CaMKII) is reported to cause diastolic SR Ca2+ leak, leading to delayed afterdepolarization (DAD), which is recognized as a major source of ectopic activity [2+ leak and spontaneous SR Ca2+ release [2+ leak and the spontaneous SR Ca2+ release. In keeping with the results of our in-vivo study, the α1-AR-specific blocker prazosin had a similar effect.To further investigate the mechanism by which both types of receptor-mediated signaling contribute to maintaining AF, we assessed the SR Caactivity ,36. Ectoactivity . β-AR ac release . Consist2+ transient in cardiomyocytes [2+, and CaMKII-mediated signaling, has been reported to induce elevation of intracellular Ca2+ concentration through SR Ca2+ release from IP3R, leading to spontaneous Ca2+ release from RYR in atrial myocytes [2+ leak that was observed in the present study may have been caused by a similar mechanism.Recent reports have documented an important role of α1-AR-mediated signaling including Gq, phospholipase C, inositol triphosphate receptor (IP3R), protein kinase C, and CaMKII in the regulation of Camyocytes –40. In amyocytes . Thus th+ current, thereby enhancing automaticity [On the other hand, it has been reported that α-AR signaling induces the activation of cardiac neurons ,43. In amaticity . These fThese findings imply that consideration of α1-AR-mediated signaling may also be important in the management of AF. Along the same lines, a recent report showed that the α, β-blocker carvedilol is more useful than the β1 selective blocker metoprolol in preventing AF after cardiac surgery .2+ transient at room temperature following the method of previous reports [A possible limitation of this study is that we measured Ca reports ,14. The 2+ leak in atrial cardiomyocytes. This model and the knowledge we have obtained through its use will be useful in establishing novel therapeutic targets and agents for the treatment of AF.In conclusion, we established an adrenergic activation-induced long-lasting AF model in mice. Using the model, we demonstrated the important role of β1- and α1-AR-mediated signaling in the maintenance of AF. In addition, we showed that not only β1-AR but also α1-AR activation are involved in the SR Ca


The cellular response to DNA double strand breaks (DSBs) is a multifaceted signaling program that centers on post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. In this review we discuss how ubiquitin and SUMO orchestrate the recognition of DSBs and explore how this influences chromatin organization. We discuss functional outcomes of this response including transcriptional silencing and how pre-existing chromatin states may control the DSB response and the maintenance of genomic stability. Every organism experiences challenges to the integrity of their DNA sequence from endogenous  and exogenous  sources. Such challenges can take the form of base mismatches and base damages or single and DSBs in the DNA backbone. Discrete molecular pathways driven by posttranslational modifications have evolved to correct each of these DNA damage types and are crucial for cellular survival and for the maintenance of genomic integrity. Perhaps the most deleterious of these lesions is the DSB as even a single unrepaired DSB can cause cell death and inaccurate repair can lead to mutations that cause cancer and other genetic diseases .The DSB response encompasses multiple post-translational modifications, including ubiquitylation (ub) and SUMOylation that primarily occur within the immediate vicinity of the DSB on chromatin and chromatin-associated proteins. Locally, this promotes DSB repair mechanisms and systemically activates cellular responses, including cell cycle checkpoints that collectively suppress genomic instability. Recent technological advances and conceptual insights have highlighted how the DSB response influences the dynamic structural organization of the nucleus. In this review we will first outline how the ubiquitin and SUMOylation systems contribute to the sensing of DSBs and then examine how these pathways affect higher order chromatin structure to maintain genetic stability.At the apex of the molecular cascade that signals double strand breaks are three phosphatidylinositol 3-kinase related kinases (PIKKs): DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR). DNA-PKcs forms an active holoenzyme, DNA-PK, with the heterodimer Ku70/80 at DNA ends and mainly contributes to DSB repair by non-homologous end-joining NHEJ; . ATR, inN-terminus to form linear chains  as the first E3 to catalyse K63 linkages at DSBs in mammals . TogetheIn addition to ubiquitin, the small ubiquitin-like modifier (SUMO) proteins have been found to impact essentially every facet of the DNA damage response by modulating protein-protein interaction and enzymatic activity . Discovein vitro and this activity is stimulated by SUMOylation of BRCA1 , localizes to SUMO-modified MDC1 at DSB sites where its ubiquitin ligase activity is required for effective RAP80-BRCA1 recruitment as well as DSB repair and effective responses to replication stress . RAP80 iof BRCA1 . Althougof BRCA1 .Recent proteomic studies have identified hundreds of damage-induced targets of both ubiquitylation and SUMOylation . The arrFigure 1). For example, moving from the nuclear interior to the periphery. We will discuss how these particular movements may influence the mechanism and fidelity of double strand break repair. These movements have been summarized in Table 1. The second category includes more localized epigenetic changes that cause transition between heterochromatic and euchromatic states. In this instance we will focus our discussion on recent insights into how these changes influence transcription near the DSBs.For the purposes of this review, we will separate chromatin reorganization into two interconnected categories. The first category includes large-scale chromatin redistributions in which the damaged locus changes its physical location within the 3D nuclear compartment ” to maintain their telomere length. Rather than activating telomerase, ALT cells utilize a homology driven mechanism to promote lengthening of telomeres . Our labDrosophila melanogaster, heterochromatic breaks are mobilized from heterochromatin to the nuclear periphery in a mechanism mediated by the STUbLs Dgrn and Rad60  and related high-throughput “C” technologies  has allowed the refinement of the chromosome territory models described above. These methods allow the interrogation of chromosome contacts that occur both within and between chromosomes. The most obvious of these contacts occur within topological associated domains (TADs) that are intrachromosomal regions of hundreds of kilobases in mammals that contain within them genes with similar expression dynamics . These Tin cis to the breaks  interactions . This dee breaks .A practical example of how differential localization can influence genome stability occurs in prostate cancer cells. When stimulated with dihydrotestosterone (DHT) TMPRSS2 gene expression is strongly induced in a manner dependent on topoisomerase II (TOP2) catalyzed DSBs that relieve torsional stresses that block transcription . These TIt is becoming clear that the dynamic organization in 3D space of the nucleus has a direct influence on genomic stability and the DSB response. As technologies advance and methods for localized induction of DSBs in mammalian cells mature it will become possible to examine how these local chromatin interactions influence, and are influenced by, the DSB response.Post-translational modifications on histones are a cornerstone of the DSB response. This has stimulated considerable interest in epigenetic marks on chromatin in the vicinity of the break site and the functional outcomes that this can entail. Recent studies from several labs have identified specific histone modifications driven by the DSB response that modulate transcription near the DSB site. We will outline some of these histone modifications in different physiological contexts and briefly discuss the functional outcomes of these events.X- and Y-chromosomes lack partners and remain largely unpaired during meiosis but remain replete with DSBs that activate a DSB response in an isolated structure called the XY-body  remain undefined . MSCI occurs at least in part due to histone modifications including H2A-ub, H3 and H4 deacetylation, and H3K9 dimethylation; these marks persist during silencing throughout meiosis even after γH2AX has been resolved  harbors 24 repeats of a stem loop that is recognized by the phage coat protein MS2 . This enam or IR . Other sir by HR . It is iD. melanogaster IR induced local decondensation of HP1a-associated heterochromatin and this was proposed to facilitate DSB repair . We found that when NHEJ was blocked nucleolar reorganization and transcriptional silencing was enhanced; this was not observed when HR was inhibited. This suggested that NHEJ was the predominant mode of DSB repair in nucleoli, which was borne out by direct repair assays at the rDNA loci. Interestingly As mentioned, translocations driven by NHEJ occur largely between chromosomes in close spatial proximity . To prevOver the last 30 years the mechanisms of the DSB response have been intensively studied and have provided an intricate model for the recognition and subsequent repair of DSBs dependent on post-translational modifications including phosphorylation, ubiquitylation, and SUMOylation. Of particular interest in the coming years will be how each of these modifications act in combination to drive accurate recognition of the breaks and repair pathway choice. SUMOylation and ubiquitylation offer a prime example of such concerted actions that are just beginning to be understood. Although technically challenging understanding how these multifaceted interactions are orchestrated is key to fully elucidating the DSB response. Recent evidence from many groups has begun to unravel these issues, and invariably they require multiple modifications rather than a single chromatin mark highlighting the importance of viewing the DSB response holistically rather than as singular distinct pathways.Technological advances in the last decade have provided the tools necessary to interrogate how the organization of the nucleus both at the global  level and at the level of the epigenome. Key consideration in this regard include how the DSB response modulates chromatin interactions during the acute phase of the DNA damage response, and if persistent DNA damage signaling alters the epigenome. Equally important will be understanding how dynamic movements in the mammalian nucleus are controlled following DNA damage. Given recent evidence that such movements are important for the generation of chromosomal translocations a molecular understanding, such as that emerging in yeast, will be a fruitful area of future study.It has also become increasingly apparent that the context  in which a DSB is induced has a significant effect on the nature of the response and outcome of repair. Several experimental approaches are now available to induce breaks within defined chromatin environments and physical locations. These systems will undoubtedly facilitate a broader understanding of the contextual aspects of the DSB response and will lead to a more unified model of nuclear organization, cell signaling, and DSB repair.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


In this paper we present our 5-year-experience with a large-scale mentoring program for undergraduate medical students at the Ludwig Maximilians-Universität Munich (LMU). We implemented a two-tiered program with a peer-mentoring concept for preclinical students and a 1:1-mentoring concept for clinical students aided by a fully automated online-based matching algorithm. Approximately 20-30% of each student cohort participates in our voluntary mentoring program. Defining ideal program evaluation strategies, recruiting mentors from beyond the academic environment and accounting for the mentoring network reality remain challenging. We conclude that a two-tiered program is well accepted by students and faculty. In addition the online-based matching seems to be effective for large-scale mentoring programs. Pinilla Severin and Pander Tanja contribute equally to the paper.http://www.wfme.org/standards/bme citited 05.02.14]. Although academic mentoring is acclaimed and perceived as a successful support tool and career catalyst, there is a lack of formal mentoring programs in many countries ..15].The peer-mentoring program was initially designed primarily for pre-clinical students but students from clinical semesters regularly participated in most peer-mentoring events as well. The driving momentum of the peer-mentoring program originates from motivated students from all semesters, so called junior mentors. The individual junior mentors are selected through a democratic selection process. After election, junior mentors are invited to a two-day introductory seminar with workshops on mentoring, leadership and team building to prepare for their role as multiplicators of the mentoring idea. Junior mentors are elected on a yearly basis and represent contact persons and mentors for their fellow students. In addition junior mentors organized a range of mentoring events.All data used in our results is generated from our website or from individual evaluation of events.Through the continuous political support of the deans of the medical school, MeCuM-Mentor was successfully integrated in the medical faculty as a formal project of the Institute for Medical Education at the LMU Munich. Currently the coordinating team consists of two full-time employees, one with a pedagogical background and another with a medical background. Additionally, four to six student assistants are involved in organizing and evaluating the peer-mentoring events throughout the academic year. The program is predominantly funded through tuition fees and through event-specific sponsors.http://www.mecum-mentor.de) which consists of both publicly accessible resources as well as password secured internal sites to administer mentor and mentee profiles and mentoring relationships. In 2013 the webpage was visited roughly 14,000 times.The central information source for all stakeholders is a webpage  as well as newsletters from the student government body and self-designed posters to announce any upcoming events or updates that are relevant in the context of mentoring and networking.In order to recruit new mentors, we introduce the program every October in all clinical departments associated with our medical school. Furthermore we organize a mentoring event (MentoringFest) once a year, which includes an introductory lecture to mentoring, a mentoring speed dating market as well as mentoring round-tables in order to facilitate participation in the mentoring program as well as meeting potential mentors from all hierarchical levels in person. Approximately 399 mentors are currently active in our program. 187 of them have still available places for mentees. On average one mentor has a formal mentoring-relationship with one to three mentees. 128 mentors are female and 271 mentors are male and they cover the full range of medical specialties  together with more specific activities like an introductory clinical skills training (‘Fit for Famulatur’) or information sessions for high-stakes exams (‘Physikumsvorbereitung’) and orientations for the clinical years (‘Klinikeinführung’) are part of a diverse peer-mentoring project portfolio . Some of those interventions have been evaluated more in-depth and have been published as reports or posters , 10], , .Considering that mentoring is a complex construct and involves a myriad of sociocultural, professional and psychological elements, it is difficult to define a single-best outcome measure that would allow deciding whether a mentoring program is successful or not . A partiAnother fundamental aspect refers to the characteristics of students who participate in a voluntary mentoring program. Based on our data, better performing students seem to be more likely to participate in a mentoring program . DespiteFurthermore mentors who are active in our program are mostly working in an academic environment. In order to provide expertise and mentorship over the full range of professional development opportunities, additional recruiting strategies in order to reach practitioners and non-clinical mentors are needed. Additionally it is not clear yet, what the ideal way to select each junior mentor cohort would be. We initially tried a democratic election, however switched to a written application due to low election participation among undergraduate students and a relatively high workload of organizing student elections.The focus of our mentoring program is on horizontal and vertical connection, sharing of experiences, building networks and facilitation of resource access. Other important aspects of mentoring, which draw on psychological counselling aspects or remediation techniques, might be underrepresented in our program since participating mentors do not have to fulfil specific criteria or go through additional training.Some researchers have moved from conceptualizing mentoring as a dyadic relationship between two persons to a network of mentors, which might be closer to the reality of mentoring , 8]. Ho. Ho8]. HThe voluntary participation of mentors and junior mentors is an essential part of our program. We are convinced that intrinsic motivation is condition as well as foundation of a programs’ success. However, formal recognition of mentoring activities might additionally increase participation rates and foster a mentoring culture within an institution or faculty.Mentoring programs do not function without continuous personal input. Despite a well-established online platform we regularly visit morning case discussions, lunch conferences and students’ lectures to inform faculty members about the benefits of participating in the program and being part of the mentoring culture at our faculty. We also established an open-door-policy in our office to provide all stakeholders of the mentoring program with a physical reference and support point.From the regularly organized peer-mentoring events with continuously high attendance rates we conclude that our junior mentor concept is successful in terms of leveraging the multiplicator potential of motivated students. Positive overall evaluation results support this observation.The two-tiered mentoring concept with an online matching algorithm seems to be a suitable solution for large-scale mentoring programs. It allows mentoring for high numbers of students by simultaneously ensuring the quality of individual support . We are At the moment there seems to be no consensus on what kind of matching process would be ideal for a mentoring program in medical school. From our experience an online matching program seems to be comparable to sympathy-based accidental and informal mentoring relationships. We assume that through our extended matching algorithm there is a potentially higher satisfaction rate as compared to random matching of mentors and mentees, although we have no data at this point to satisfactorily test this hypothesis. We suggest that future studies use controlled trials to compare measurable outcomes, such as mentoring satisfaction, frequency of interaction between mentor and mentee and duration of mentoring relationship in order to provide better recommendations for how the matching process should be designed.Depending on the financing and hierarchical structure of a mentoring program it is important to effectively collaborate with different departments and student association bodies in order to have the necessary political support from a long-term perspective. Official support from decision-makers and stakeholders is also crucial in this context. We found the support from well-regarded active and retired professors and deans as catalyzing for launching the mentoring program as well as for initiating new mentoring activities.Finally, an effective and efficient mentoring program can also function as a catalyst for medical education innovations. The aggregated expertise and experience in organizing events from small informal gatherings to communication and clinical skills courses as well as student research conferences can serve as a continuous source to foster educational innovation and scholarship.The five-year experience with our mentoring program shows that a large-scale mentoring program could be successfully integrated in the medical faculty of the LMU Munich. A two-tiered mentoring concept, with individual mentors for clinical semesters and junior mentors, works successfully for faculties with high numbers of students. Intrinsic motivation of the mentors and formal supportive structures are crucial. The presented voluntary mentoring concept is being used by roughly every fourth student of our faculty and should be transferable to other faculties and disciplines. Longitudinal and multi-centered research projects are needed to better estimate the effects of mentoring relationships on individual career paths and career satisfaction as well as on personal and professional network development and work-life-balance. Innovations in communication technology and use of social media might also change the scope of mentoring activities and provide additional opportunities to effectively mentor medical students.We would like to thank Sylvère Störmann and Stefan Galster for their outstanding support with programing the web-based components of MeCuM-Mentor.The authors declare that they have no competing interests.


P. falciparum malaria. The emergence of P. falciparum resistance to artemisinin has recently developed in Southeast Asia. Little data are available on the association between pyronaridine susceptibility and polymorphisms in genes involved in antimalarial drug resistance. The objective of the present study was to investigate the association between ex vivo responses to pyronaridine and the K76T mutation in the pfcrt gene in P. falciparum isolates.The pyronaridine-artesunate combination is one of the most recent oral artemisinin-based therapeutic combinations (ACTs) recommended for the treatment of uncomplicated ex vivo susceptibility to pyronaridine was performed on 296 P. falciparum isolates using a standard 42-h 3H-hypoxanthine uptake inhibition method. The K76T mutation was also investigated.The assessment of 50 (inhibitory concentration 50 %) ranged from 0.55 to 80.0 nM. Ex vivo responses to pyronaridine were significantly associated with the K76T mutation . The reduced susceptibility to pyronaridine, defined as IC50 > 60 nM, was significantly associated with the K76T mutation . Using a Bayesian mixture modelling approach, the pyronaridine IC50 were classified into three components: component A (IC50 median 15.9 nM), component B (IC50 median 34.2 nM) and component C (IC50 median 63.3 nM). The K76T mutation was represented in 46.3 % of the isolates in component A, 47.2 % of the isolates in component B and 73.3 % of the isolates in component C .The pyronaridine ICex vivo reduced susceptibility to pyronaridine, i.e., IC50 > 60 nM, associated with the K76T mutation.These results showed the  P. falciparum and P. vivax malaria ). The in vitro resistance to chloroquine, defined as IC50 > 100 nM, was also significantly associated with the K76T mutation . The odds ratio for reduced susceptibility to chloroquine associated with the K76T mutation was 96.4 (95 % CI [41.8–244.8]). Using Bayesian mixture modelling, the 296 pyronaridine IC50values were classified into three components: component A (IC50 median 15.9 nM), component B (IC50 median 34.2 nM) and component C (IC50 median 63.3 nM) . The proportion of isolates in each group was 59.8 % for component A, 30.1 % for component B and 10.1 % for component C. The K76T mutation represented 46.3 % of the isolates in component A, 47.2 % of the isolates in component B and 73.3 % of the isolates in component C .The pyronaridine IC nM Fig. . The geoin vitro . The redex vivo reduced susceptibility to pyronaridine, i.e., IC50 > 60 nM, was associated with the K76T mutation in the pfcrt gene. These ex vivo results are in contrast with the in vitro results in 23 P. falciparum strains in which the in vitro to pyronaridine were not associated with pfcrt polymorphisms [50 of 6 versus 20 nM). However, this difference was not significant  [The results of the present study showed that orphisms . However > 0.05) . The oddpfcrt polymorphism in isolates from Africa and Asia [pfmdr6 [In contrast with piperaquine, in which its in vitro responses are not associated with and Asia , 17, 18  [pfmdr6 , pyronarP. berghei parasites, while artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine and dihydroartemisinin-piperaquine are not effective [P. vivax malaria [Pyronaridine-artesunate successfully treats artemisinin-resistant ffective . Pyronarffective –4. Pyronffective . In addi malaria . PyronarAccording to the French legislation, bio-banking and secondary use for scientific purposes of human clinical samples are possible as long as the corresponding patients are informed and have not indicated any objections. This requirement was fulfilled here since information is given to every patient through a hospital notice entitled “Information for Patients,” and no immediate or delayed patient opposition was reported by the hospital clinicians to the French Malaria Reference Center. Moreover, samples received at the French Malaria Reference Center were registered and declared for research purposes as a bio-bank for the French National Institute of Health Survey. No institutional review board approval is required according to French legislation .


An approach to investigate brain function/dysfunction is to simulate neuron circuits on a computer. A problem, however, is that detailed neuron descriptions are computationally expensive and this handicaps the pursuit of realistic network investigations, where many neurons need to be simulated.We confront this issue; we employ a novel reduction algorithm to produce a 2 compartment model of the cerebellar Purkinje neuron from a previously published, 1089 compartment model. It runs more than 400 times faster and retains the electrical behavior of the full model. So, it is more suitable for inclusion in large network models, where computational power is a limiting issue. We show the utility of this reduced model by demonstrating that it can replicate the full model’s response to alcohol, which can in turn reproduce experimental recordings from Purkinje neurons following alcohol application.We show that alcohol may modulate Purkinje neuron firing by an inhibition of their sodium-potassium pumps. We suggest that this action, upon cerebellar Purkinje neurons, is how alcohol ingestion can corrupt motor co-ordination. In this way, we relate events on the molecular scale to the level of behavior. At present, there are two minimally interacting levels of investigation in computational neuroscience. In the first, researchers use Hodgkin-Huxley models of currents  and compIn vitro, within cerebellar slices, Purkinje neurons can spontaneously fire action potentials in a repeating trimodal pattern that consists of tonic spiking, bursting and quiescence ) for the extrusion of every doubly positive Ca2+ ion (1*[+2])  OR . These entities are voltage  dependent. The latter set can give the former set through the relations:The voltage  dependence of a Hodgkin-Huxley (H-H) current  can be eK is the reversal potential for K+ (initiated at -88 mV), ENa is the reversal potential for Na+ (initiated at +70 mV), ECa is the reversal potential for Ca2+ , EL is the reversal potential for the Leak current (-70 mV), Eh is the reversal potential for the hyperpolarisation activated cation current (-30 mV), Intracellular Ca2+ concentration is initiated at the NEURON default of 5*10−5 mM; Extracellular Ca2+ concentration is initiated at the NEURON default of 2 mM. The somatic membrane voltage (V) is initiated at the NEURON default of -65 mV.The soma is a cylinder . EK_fast), moderately TEA sensitive (IK_mid) and TEA insensitive (IK_slow) voltage-gated K+ currents, a BK voltage-and-Ca2+-gated K+ current (IBK), a resurgent Na+ current (INa-R), a P-type Ca2+ current (ICaP), a hyperpolarization activated cation current (IH), a leak current (IL), a SK Ca2+-gated K+ current (ISK) and an intracellular Ca2+ dynamics abstraction. The soma has two Na+/K+ pump descriptions (Ispump and ispump) and a Na+/Ca2+ exchanger mechanism Isex.The soma has highly TEA sensitive  equation:Ca2+ is 5*10−5cm/sec, [Ca2+]i = 100 nM, [Ca2+]o = 2 mM, T = 295 K, F is the Faraday constant and R is the gas constant. [Ca2+]i and [Ca2+]o are fixed constants, as seen by this equation – it does not access the changing value of [Ca2+]i as set by the intracellular Ca2+ equations (given later).P2+] is calculated for the intracellular space within 100 nm of the membrane. [Ca2+] changes as ICa2+  brings Ca2+ into this space and as Ca2+ leaves by diffusion to the bulk cytoplasm. The diffusion rate constant,β, is set to 1/msec.[Ca2+] at time step, t: was constrained to not fall below 100 nM by coding of the form:−1):This current is described by a Markov scheme, shown in Figure +/K+ pump  transports 3 Na+ out (ispump_Na) for every 2 K+ in (ispump_K). It has a fixed voltage (V) dependency and an exponential relation to intracellular Na+ concentration ([Na+]i). The affinity constant for Na+, KNa = 40 mM.The somatic Na+/Ca2+ exchanger mechanism (Eq. +/K+ pump mechanism (Eq. +/Ca2+ exchanger current (Isex_net) is net depolarizing (-1), inwardly passing 3 singly positive Na+ ions (3*[+1]) for the extrusion of every doubly positive Ca2+ ion (1*[+2]). By contrast, the Na+/K+ pump current (Ispump_net) is net hyperpolarizing (+1) in its transport of 3 Na+ out (3*[+1]) for every 2 K+ in (2*[+1]). The exchanger density is gsex; the pump density is gspump.The soma has a simple Nanism Eq.  and a sinism Eq. . The Na++/K+ pump current of Eq. +/Ca2+ exchanger current of Eq. slight mismatch  . The second term on the Right Hand Side (RHS) of Eq. + out of the soma compartment, along the longitudinal distance (x). The effects of this term are fairly negligible and it could be dropped to quicken simulation speeds. INa_net is the difference between Na+ current flowing into the soma  and Na+ current pumped out of the soma by the Na+/K+ pump (INa_out), lagged by parameter τ = 5 s. Intracellular Na+ stimulates the Na+/K+ pump and this lag τ accounts for the duration of sodium’s diffusion from channels to pumps. ispump_Na and Ispump_Na are Na+ currents produced by the pumping action of the Na+/K+ pump at the soma, set by Eq. K is the reversal potential for K+ , ENa is the reversal potential for Na+ , ECa is the reversal potential for Ca2+ , EL is the reversal potential for the Leak current (-80 mV), Eh is the reversal potential for the hyperpolarisation activated cation current (-32.9 mV). Intracellular Ca2+ concentration is initiated at 4*10−5 mM; Extracellular Ca2+ concentration is initiated at 2.4 mM. Extracellular Na+ concentration is set by the NEURON default value; +140 mM.The dendrite is a cylinder . EH); T-type (ICaT), Class-E (ICaE) and P-type (ICaP) voltage-gated Ca2+ currents; a leak current (IL); A-type (IKA), D-type (IKD), M-type (IKM), Delayed Rectifier (IDR) and Kv1.2 (IKv1.2) voltage-gated K+ currents; BK (IBK) and K2 (IK2) type voltage-and-Ca2+-gated K+ currents and an intracellular Ca2+ dynamics abstraction. The dendrite has two Na+/K+ pump descriptions (Idpump and idpump) and a Na+/Ca2+ exchanger mechanism Idex.All the mechanisms in the dendrite are distinct from those in the soma. The dendrite has hyperpolarization activated cation current .T is temperature in degrees centigrade (36).T is temperature in degrees centigrade (36).T is temperature in degrees centigrade (36).T is temperature in degrees centigrade (36).T is temperature in degrees centigrade (36).T is temperature in degrees centigrade (36).These equations are based upon the NEURON code of , as oppoWhere fabs(x) returns the absolute value of a floating point number; the absolute value of its argument (|x|).T is temperature in degrees centigrade (36).These equations are based upon the NEURON code of , as oppoThese equations are based upon the NEURON code of , as oppo2+]i is the intracellular Ca2+ concentration in a supra-membrane shell of depth = 0.1 μm, F is the Faraday constant, 2+ membrane current , kt = 4*10−5 mM/ms, kd = 4*10−5 mM, taur = 2 ms and y = 4*10−5 mM.+/K+ pump  has a 3Na+:2 K+ stoichiometry, no voltage dependency and a hyperbolic relation to extracellular K+ concentration ([K+]o.The dendritic Na+/Ca2+ exchanger mechanism (Eq. +/K+ pump mechanism (Eq. +/Ca2+ exchanger current is net depolarizing (-1), inwardly passing 3 singly positive Na+ ions (3*[+1]) for the extrusion of every doubly positive Ca2+ ion (1*[+2]). By contrast, the Na+/K+ pump current is net hyperpolarizing (+1) in its transport of 3 Na+ out (3*[+1]) for every 2 K+ in (2*[+1]).The dendrite has a simple Nanism Eq.  and a sinism Eq. . The Na+dex and gdpump are Na+/Ca2+ exchanger and Na+/K+ pump membrane current densities (respectively) in the dendrite and their equality at 0.0021 mA/cm2 ensures an electrical counterbalance So, the model dendrite has a Na+/Ca2+ exchanger current and an electrically counterbalancing Na+/K+ pump current:g+ concentration ([K+]o) to the dendrite is initiated at 2 mM and then changes in time, t, according to the relationship:F is the Faraday constant, wid is the thickness of an extracellular region around the compartment that K+ accumulates in (70*10−3 μm), Q is a K+ accumulation factor (0.0119) and IK_net : we observe that the shorter the length of the dendrite compartment, the higher the dendritic spiking frequency and the less spikes per burst. However, we are happy with the present degree of concordance achieved. As we shall demonstrate, our reduced model is a powerful tool for capturing and predicting behavior in the full model.The 2 compartment model can replicate the behavior of the full, 1089 compartment model; it can replicate the cerebellar Purkinje cell’s trimodal pattern of spontaneous activity Figure . The cavOn an Intel core PC (i5), the 2 compartment model took 1.11 seconds of CPU time for 1 second of simulation. So, simulations with this model run at real time (approximately). This is ~414* faster than the 1089 compartment model.+/K+ pump equations ; it is used to prevent negative values of  from occurring.The model has four different Naquations . They eaNa  and simulating it for 120 model seconds was not a problem. The full model, however, could not be simulated over this time frame, on these means, within a reasonable time span. We found that this is because the real time taken to simulate a given unit of model time increases to larger values with longer simulations. We simulated the full model for 20 seconds and using the real time taken for this simulation, we extrapolated that a simulation of 120 seconds would take at least 3 days; if not a lot more. The full model was uploaded to the Neuroscience Gateway Portal ; RRID: nline Eq.  that pro and simuOur detailed Purkinje neuron model was run serially on the Trestles supercomputer but we anticipate that our source code could be changed in the future such that it could be run in a parallel fashion. Recently, a parallel computation method has been shown to be viable for single neuron models ,48 and tHere we shall demonstrate that the 2 compartment, reduced model can be used as a proxy for tuning the detailed, morphologically faithful model. We show that the reduced model can be used to find parameter values that will permit the full model to replicate new behaviour; that observed experimentally for cerebellar Purkinje neurons in mice that have a BK channel genetic knockout ,32.2+ spikes in the dendrites are repolarised by the BK potassium current. These spikes travel to the soma and generate the bursting mode of the trimodal firing pattern  at


Conscious mice were used in order to preserve normal mean arterial blood pressure (MAP) and sympathetic nerve activity (SNA). ExMLCK mice, which express a genetically-encoded smooth muscle-specific FRET-based Ca2+ indicator, were equipped with blood pressure telemetry and immobilized for imaging. MAP was 101 ± 4 mmHg in conscious restrained mice, similar to the freely mobile state (107 ± 3 mmHg). Oscillatory vasomotion or irregular contractions were observed in most arterioles (71%), with the greatest oscillatory frequency observed at 0.25 s−1. In a typical arteriole with an average diameter of ~35 μm, oscillatory vasomotion of a 5–6 μm magnitude was accompanied by nearly uniform [Ca2+] oscillations from ~0.1 to 0.5 μM, with maximum [Ca2+] occurring immediately before the rapid decrease in diameter. Very rapid, spatially uniform “Ca2+ flashes” were also observed but not asynchronous propagating Ca2+ waves. In contrast, vasomotion and dynamic Ca2+ signals were rarely observed in ear arterioles of anesthetized exMLCK biosensor mice. Hexamethonium  caused a fall in MAP to 74 ± 4 mmHg, arteriolar vasodilation, and abolition of vasomotion and synchronous Ca2+ transients.Two-photon fluorescence microscopy and conscious, restrained optical biosensor mice were used to study smooth muscle CaSummary: MAP and heart rate (HR) were normal during high-resolution Ca2+ imaging of conscious, restrained mice. SNA induced continuous vasomotion and irregular vasoconstrictions via spatially uniform Ca2+ signaling within the arterial wall. FRET-based biosensor mice and two-photon imaging provided the first measurements of [Ca2+] in vascular smooth muscle cells in arterioles of conscious animals. Two major considerations motivated this work: (1) Ca2+ signaling in arteries under physiological conditions with normal arterial blood pressure and intrinsic regulation via circulating hormones and the autonomic nervous system had never before been observed. (2) Studies of Ca2+ signaling in experimental hypertension have been complicated by the effects of anesthesia to reduce arterial blood pressure probably by reducing sympathetic nerve activity (SNA) to arteries. As a consequence, genuinely representative in vivo measurements of intracellular [Ca2+] did not exist.Here we report the first non-invasive measurements of intracellular smooth muscle [Ca2+] in arterioles of intact ears of anesthetized hypertensive mice  the use of transgenic optical biosensor mice, that express the genetically encoded FRET-based CaOne anesthetic cocktail involving a mixture of ketamine, medetomidine, and atropine is able to prevent the reduction of MAP typically observed in anesthetized mice; however it strongly reduces heart rate (HR)  , which expresses a FRET-based CaArterial blood pressure was recorded via telemetric pressure transducers  implanted in the right carotid artery. A period of recovery lasting 7–10 days was provided before animals were utilized for experiments.Mice were anesthetized using 2% isoflurane in 100% oxygen. The scalp was trimmed using hair clippers, cleaned with iodine and alcohol, and the dorsal skull surface was removed with surgical scissors. Sterile cotton swabs were used to scrape away the periosteum, clean and dry the skull surface. A 2.5 cm long threaded stainless steel bar was secured in a horizontal position above the skull, extending toward the non-experimental ear using 3M ESPE dental cement (Dentsply). Animals recovered for 3 days before experimentation. During experiments, the animals' heads were fixed in position by attaching the threaded bar to an anchored stage, their bodies were immobilized inside a custom-made conical tube, and their depilated ears were positioned on a flat horizontal silicone platform as previously detailed  at least 3 days prior to imaging. Mice were lightly anesthetized (1.5% isoflurane) and then placed in a prone position on a temperature-controlled platform set to maintain core temperature at 37°C, which was monitored via rectal thermoprobe. The ventral surface of the depilated ear was affixed to a silicone platform with adhesive tape, situating the dorsal surface of the ear for imaging Figure . This arA Zeiss LSM 710 NLO microscope was utilized for two-photon microscopy, equipped with a femtosecond pulsed near infrared (IR) laser . The frame rate and pixel size of the bidirectional scanning varied for each experiment, but ranged from 2 to 5 Hz and 0.59 to 0.83 μm/pixel, respectively. The microscope was enclosed in a light-tight enclosure and room lights were turned off to eliminate background signals. The CFP moiety of exMLCK was excitation at 820 nm. Emission light received IR filtration before being separated into two channels (CFP and YFP). CFP and YFP emission was band pass filtered from 460 to 500 nm and 520 to 560 nm, respectively. Two binary GaAsP photodetectors within the Zeiss LSM BiG module detected fluorescence emission. The gains of the two detector channels were held constant for all experiments at levels optimizing exMLCK fluorescence. The configuration settings of the detectors were reproduced during calibration experiments, which determined the maximum and minimum FRET ratios obtainable from our microscope  was calculated as Y = (R – Rmin)/(Rmax – Rmin). The Calcium concentration wherein Y = 0.5 represents the EC50. The fluorescence ratios Rmax and Rmin, respectively, represent 100 and 0% fractional occupancy of exMLCK by Ca2+/Calmodulin. Free [Ca2+] is calculated from Y, using the Hill equation, as [Ca2+] = [(Y·ECn50)/(1.0 – Y)]1/n. The relationship between free [Ca2+] and exMLCK FRET ratio, R, as measured in α-toxin permeabilized mesenteric small arteries, is well-fitted by the Hill equation, with an EC50 (KA) of 0.892 μM  and a Hill Coefficient (n) of 1.4  was inserted in the abdominal cavity and anchored to the skin with adhesive tape to intraperitoneally deliver hexamethonium 30 μg/g BW  dissolved in sterile saline (0.9% NaCl).n denotes the number of animals. Comparisons of data were made using Student's paired or unpaired t-test, as appropriate. Differences were considered significant at P < 0.05. Image processing was via custom software routines using IDL . Blood pressure measurements were analyzed with DSI software.Data are expressed as means ± s.e.m.; n = 4) was not significantly different from that in the conscious, unrestrained animal (107 ± 3 mmHg) . Mice periodically attempted activity ~3–4 times per minute, and this was associated with transient increases in MAP, as occurs in the normal basal state.Mice typically required 7–10 days to regain normal MAP following surgical implantation of telemetric blood pressure transducers and head restraints. MAP recordings began as the animal was engaging in normal behavior within its housing cage. Recordings continued into isoflurane induction while the animal was affixed to the restraint device and also during restoration of consciousness  while restrained Figure . Because) Figure . In cont2+] were present in 71% (n = 7) of the arterioles observed in the conscious, restrained state . For the arteriole illustrated in Figure 2+] oscillations from ~0.1 to ~0.5 μM  of arterial diameter for these three types of vasomotion . Thus, peak [Ca2+] is immediately followed by the maximum rate of vasoconstriction. With greater positive and negative lags, strong correlations occurred only with regular oscillating vasomotion, as would be expected for repetitive, stereotypical events. Irregular oscillating vasomotion and spontaneous vasoconstrictions exhibited no additional strong correlations other than the primary 0.3 s lag.Whether the oscillating vasomotion was highly regular Figure , irreguln Figure . In all 2+] measurements in a conscious animal , acting on α1-adrenergic receptors (α1-AR)  activated by neurally released ATP. Such Ca2+ transients may thus reflect mainly Ca2+ entry through voltage-gated Ca2+ channels. Thus, smooth muscle membrane potential appears to be critical to control of contraction of these arterioles in vivo.In isolated arteries, stimulation of sympathetic nerve terminals within the arterial wall elicits several types of Ca2+ signaling drives synchronous contractile activation in arterioles in conscious animals. Although the physiological significance of such vasomotion remains unclear, it is thought likely to enhance tissue dialysis (for review, see Aalkjaer et al., Clearly, highly synchronized Ca2+ waves are readily produced by exposure to α1-AR agonists. It has also been suggested that such Ca2+ waves are involved in myogenic tone (Mufti et al., 2+ waves were not seen in any blood vessel in the present study. Such Ca2+ signals were also not detected in our previous studies, either in cremaster muscle arterioles (Mauban et al., 2+ waves observed in vitro remain elementary signals and fail to emerge as a phenomenon in a fully intact physiological system.In isolated pressurized arteries, asynchronous propagating Ca2+ signaling in the smooth muscle cells of blood vessels in the ears of restrained conscious mice is readily achieved through the use of optical biosensor mice and two-photon microscopy. Arterioles and small arteries of the mouse ear are under tonic control by the sympathetic nervous system, and the predominant mode of contractile activation seems to be spatially uniform Ca2+ signaling, insofar as can be observed with exMLCK. Although both myogenic tone, and sympathetic neurogenic contractions of isolated arteries can involve asynchronous propagating Ca2+ waves in individual cells, such Ca2+ signals were never observed in the present study. These results reinforce the notion that synchronization of Ca2+ signaling in vivo is important for physiological function.In summary, high resolution imaging of CaThe 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.


Furthermore, we find that CA2 dendritic geometry contributes to the efficient coupling of dendritic Na+ spikes to AP output. These results provide a striking example of how dendritic spikes enable direct cortical inputs to overcome unfavorable distal synaptic locale to trigger axonal AP output and thereby enable efficient cortico-hippocampal information flow.Synaptic inputs from different brain areas are often targeted to distinct regions of neuronal dendritic arbors. Inputs to proximal dendrites usually produce large somatic EPSPs that efficiently trigger action potential (AP) output, whereas inputs to distal dendrites are greatly attenuated and may largely modulate AP output. In contrast to most other cortical and hippocampal neurons, hippocampal CA2 pyramidal neurons show unusually strong excitation by their distal dendritic inputs from entorhinal cortex (EC). In this study, we demonstrate that the ability of these EC inputs to drive CA2 AP output requires the firing of local dendritic NaDOI:http://dx.doi.org/10.7554/eLife.04551.001 Cells called neurons carry information—in the form of electrical signals—around the brain. These cells connect to each other in complex networks and each neuron is able to form junctions, or synapses, with many neighbors. In a neuron, small electrical signals start from synapses at the tips of branched structures called dendrites. From there, these signals travel to the cell body of the neuron to activate a larger electrical signal—called an action potential—that travels along a long tail-like extension, called the axon, to reach synapses with other neurons.+ spikes. Although they were first recorded over 40 years ago, it is not clear how important the Na+ spikes are for triggering action potentials.In the dendrites, the small electrical signals can be amplified by rapid changes in the concentration of sodium ions, known as Na+ spikes are able to travel from the dendrites to the cell body of these neurons, where they are required to trigger action potentials. However, this is not the case for other neurons in the hippocampus, where the Na+ spikes are very weak by the time they reach the cell body.In this study, Sun et al. studied a type of neuron in the hippocampus called CA2 pyramidal neurons, which are involved in social memory and aggression. Unlike most other neurons in this region, CA2 neurons are strongly activated by signals from a neighboring region of the brain called the entorhinal cortex. The experiments show that Na+ spikes travel to the cell body. The next major challenge is to understand the role of the Na+ spikes in social memory and other complex behaviors that are controlled by CA2 neurons.Sun et al. used a computational modeling technique to compare the different types of neurons in the hippocampus. The dendrites of these cells have different branching patterns and shapes, and the model suggests that this may explain the differences in how well the NaDOI:http://dx.doi.org/10.7554/eLife.04551.002 Over th in vivo . One proitiation . Howeveratic APs . Indeed,atic APs . Thus, wasticity , it is lasticity . In this+ spikes that are necessary to trigger axonal AP output in response to a single or a burst of PP stimuli. Furthermore, these spikes can overcome strong inhibition to elicit AP output. In contrast, activation of PP inputs to distal dendrites of CA1 PNs with a single stimulus fail to elicit dendritic spikes or somatic APs. Through computational modeling based on morphological reconstructions of CA2 and CA1 PNs, we find that the distinct dendritic geometry of CA2 PNs contributes to the ability of CA2 neurons to efficiently couple dendritic Na+ spikes to AP output. Thus our data provide a striking example of how dendritic structure and functional properties control the ability of dendritic Na+ spikes to couple synaptic input at distal dendrites to axonal AP initiation. In this manner, CA2 PN dendritic Na+ spikes ensure the efficient propagation of cortical information through the EC → CA2 → CA1 disynaptic pathway.Hippocampal CA2 PNs represent a relatively small population of cells interspersed between CA3 and CA1. Nonetheless, these neurons have recently been shown to be crucial for social memory  and aggrOur laboratory found that CA2 PNs receive strong excitatory inputs from EC and fire APs with high probability in response to a brief burst of stimuli delivered to the EC PP axons (5 pulses at 100 Hz). In contrast, the same PP stimuli generate a smaller synaptic response in CA1 PNs that is usually insufficient to elicit spike output . In thisWe previously surmised that CA2 synaptic responses elicited by a focal stimulating electrode placed in SLM of CA1 were generated by activation of EC inputs from LIII pyramidal neurons, because their axons form dense projections throughout the CA1 SLM layer . HoweverIn the vast majority of CA2 PNs tested (35/42 cells), a single electrical stimulus (up to 48 V) delivered to the PP was sufficient to elicit a large, fast AP that was >80 mV in amplitude when recorded in the CA2 soma . The cumTo rule out the possibility that CA2 spike firing was an artifact of washout caused by whole-cell recording and to measure the collective activity of a population of neurons, we measured the extracellular population spike (PS) in response to PP stimulation using an extracellular field recording electrode placed in stratum pyramidale (SP) of the CA1, CA2, or CA3b region . ConsistThe efficient triggering of action potential output from CA2 PNs in response to PP stimulation is surprising given that the largest EPSP observed in the CA2 PN soma was ∼11 mV below the voltage threshold of AP output in response to somatic current injection . Thus, e+ spikes reported in other studies  . AlthougAs the rapidly rising spikelets were observed in only a subset of our recordings, we wondered whether they were a consistent feature of CA2 responses to PP stimuli but were normally masked by the much larger somatic AP e.g. . In agreWe next asked whether dendritic spikes were also a feature of PP-evoked action potentials in CA1 PNs. As a single PP stimulus was ineffective in triggering a somatic action potential in CA1 PNs, we used a 50-Hz burst of 5 strong PP stimuli to evoke somatic spiking. In marked contrast with our results with CA2 PNs, neither our somatic voltage recordings nor phase-plane plots showed evidence of a spikelet . MoreoveTo examine more directly whether dendritic spikes consistently underlie the CA2 PN somatic AP, we prevented AP firing by injecting negative current to hyperpolarize the somatic membrane to −82.5 ± 2.8 mV (n = 10) . StrikinAs a second means of recording spikelets in the absence of somatic APs, we locally applied tetrodotoxin  to the soma of CA1 or CA2 PNs while maintaining the neurons at their initial resting potential . In CA2 + spikes. Indeed, this manipulation fully blocked the ability of single PP stimuli to elicit somatic APs   and are necessary for somatic AP initiation with a single PP stimulus.Next, we asked whether PP-driven dendritic spikes are necessary to generate AP output in CA2 PNs. As discussed above, one indication for the necessity of dendritic spikes is our finding that the threshold to fire a somatic AP with PP stimulation is negative to the threshold with somatic current injection . To direatic APs , n = 6.  −73 mV) , below t −73 mV) . These r+ spikes were necessary for AP output in CA2 PNs with either single or bursts of PP input.In awake-behaving animals, EC neurons often fire in high-frequency bursts rather than isolated single APs . It is t+ spikes can influence the timing of AP output and thus have been proposed to contribute to temporal coding  cell body layers in response to a burst of PP stimuli (5 pulses at 50 Hz). In CA1, a measurable PS was never observed following the first PP stimulus and was only detected during the later stimuli in a burst, resulting in a latency to firing of 20 ms or more  and in the SP layer of CA1, CA2, or CA3 regions . CA2 denng sites . These rIh, and lack of AP adaptation  to facilitate dendritic excitation. However, under physiological conditions, the distal dendrites of CA2 PNs receive powerful inhibition that can significantly influence their excitation by the EC inputs. We thus asked whether dendritic NaP output .+ spikes or APs). Given that the resting potential of CA2 PNs was −75.6 ± 0.7 mV (n = 23) and the AP threshold with somatic current injection was −44.3 ± 0.8 mV (n = 17), this level of depolarization is very far from threshold for eliciting somatic APs. Surprisingly, however, extracellular field recordings showed that a single PP stimulus was, in fact, able to evoke a PS in the CA2 cell body layer even with inhibition present  amplitude as a function of PP stimulus strength in the presence or the absence of GABAR antagonists. For stimulus strengths above 12 V, the PP PSP was significantly enhanced by the GABAR antagonists, indicating that PP stimulation did indeed elicit strong inhibition . At an i present . As expe present .10.7554/Somatic whole-cell recordings confirmed that a single PP stimulus was able to elicit AP firing when inhibition was present, although with a reduced probability compared to when inhibition was blocked . With in+ spikes), or sub-threshold PSPs , slowly rising (dV/dt < 10 V/s) spikelets , delayed rectifier and A-type K+ conductances (GKdr and GKA), and a hyperpolarization-activated cation conductance, Ih .We lowered the + spikes that propagated to the soma to trigger AP output. By contrast, in the CA1 PN model, although a similar level of distal synaptic stimulation was able to trigger local Na+ spikes at the apical tufts, these spikes propagated poorly to the soma and failed to initiate AP output  was necessary to evoke a somatic AP . The mul+ spikes, driven by cortical inputs to the distal dendrites of CA2 PNs, propagate to the soma and are required to trigger axonal AP output. The dendritic Na+ spikes thus enable the cortical inputs to overcome their unfavorable distal dendritic location and effectively propagate cortical information by shortening spike latency and overcoming powerful inhibition. Our data further show that dendritic Na+ spikes enhance the temporal precision of CA2 PN AP output and are likely crucial for the function of the EC → CA2 → CA1 disynaptic pathway  for 20–30 min and then kept at room temperature for at least 1.5 hr before transfer to the recording chamber. Cutting and recording solutions were both saturated with 95% O2 and 5% CO2 (pH 7.4). All electrophysiological recording experiments were performed at 31–32°C. For some experiments, a cut was made between CA2 and CA3 regions.Transverse hippocampal slices were prepared from 5- to 8-week old C57BL/6J male mice from the Jackson Laboratory, as described previously . In brie2GTP, 10 Na2 phosphocreatine . Series resistance and resting membrane potential were monitored throughout each experiment. Neurons with series resistance >25 MΩ (somatic) or >50 MΩ (dendritic) were excluded from analysis. Neurons with resting potential more positive than −60 mV were also rejected from analysis. Synaptic potentials, dendritic spikes, and AP outputs were evoked by monopolar stimulation with a patch pipette filled with 1 M NaCl and located in SLM of the CA1 region (∼50 μm from CA2 region). CA2 PNs were identified based on a number of electrophysiological properties as described previously  containing (in mM): 135 K gluconate, 5 KCl, 0.1 EGTA-Na, 10 HEPES, 2 NaCl, 5 Mg ATP, 0.4 Naeviously , includiA and GABAB antagonists .Neurons were held at −70 to −73 mV for input–output curves and for examining the effect of PP stimulation on dendritic spikes and AP output. Resting membrane potential was measured immediately upon break-in. Except for the experiment shown in Extracellular field potentials were recorded with glass patch pipettes containing 1 M NaCl. The recording pipettes were placed in the pyramidal layer or various locations along SR in CA1, CA2, or CA3 fields. Field responses were evoked using a stimulating electrode placed in SLM of the CA1 field (∼50 μm from the border with CA2). Except for the experiments shown in Neurons were filled with biocytin using whole-cell patch recordings that were held for >15 min to allow for diffusion of biocytin. The slices were fixed and kept overnight in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 4°C. The slices were then rinsed five times for five minutes per rinse in 0.1 M PB and were treated with 0.3–1% hydrogen peroxide in 0.1 M PB for 30–40 min. After three rinses, slices were treated with 2% Avidin–Biotin-Peroxidase Complex  for 1–2 days. Each slice was then developed with 0.05–0.07% 3,3′-diaminobenzidine tetrahydrochloride (DAB) and 0.005% hydrogen peroxide until the slice turned light brown. Subsequently, slices were rinsed in PB several times and processed through increasing concentrations of glycerol and then embedded in mounting media .Neurons with robust staining of the dendritic tree were reconstructed using Neurolucida software . The neurons were viewed with a 63x oil objective on a Zeiss upright light microscope. Whole-cell reconstructions included the soma and dendritic branches and shafts, but not dendritic spines.m = 40,000 Ω cm2, Cm = 0.75 μF/cm2, Ri = 150 Ω cm). To account for spines, Cm of the dendritic compartments was multiplied by a spine scale factor and their Rm was divided by the same factor. In the CA1 PN model, we used a spine scale factor of 2 in compartments 150 μm beyond the soma, whereas in the CA2 PN model we used spine scale factors of 2 or 3 for compartments below or beyond 150 μm from the soma, respectively. These spine scale factor values were chosen to match the membrane time constant and input resistance values of the models to our experimental values.Three dimensional whole neuron reconstructions, including dendritic diameters and lengths, were imported into the NEURON simulation environment . To buil+ conductance (GNa), a delayed rectifier K+ conductance (GKdr), an A-type K+ conductance (GKA), and a hyperpolarization-activated cation conductance (Gh). The biophysical parameters of GNa, GKdr, GKA, and Gh were implemented, as described previously  with a reversal potential of 0 mV with specific synaptic conductance values described below. For distal synaptic activation, excitatory synapses (0.0002 µS per synapse) were distributed randomly onto the distal dendritic arbor (>400 µm from the soma). Weak synaptic stimulation was performed by activating ∼75 synapses randomly, whereas in strong synaptic stimulation ∼1000 synapses were activated randomly in both models. For the branch model  and were acquired using AxoGraph X software . Data analysis was performed using Igor Pro , AxoGraph X, and Excel . Phase-plane plot and dV/dt values were obtained with the build-in programs in AxoGraph X. AP threshold was defined as the somatic voltage at which dV/dt exceeded 10 V/s or 50 V/s. To determine the EPSP amplitude when dendritic spikes were present e.g. , EPSP am review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , Gary Westbrook (Reviewing editor), and 2 reviewers.Thank you for sending your work entitled “Dendritic NaThe Reviewing editor and the reviewers discussed their comments before we reached a decision, and assembled the following comments to help you prepare a revised submission. The reviewers found the experiments to be convincing and the findings to be very significant, demonstrating a pyramidal neuron with reliably propagating D spikes. However, both reviewers had concerns about the conclusions regarding morphology and aspects of the modeling that require your attention.Major comments:+ spiking that can facilitate somatic action potential initiation including in physiological-like conditions (even in the presence of GABA-mediated inhibition). However, the authors' conjecture that morphology alone seems sufficient to explain efficiently propagation of dendritic spikes in CA2 PCs is rather simplistic and relatively untested. Although the authors attribute the differences between CA1 and CA2 cells to morphology, they actually have differences in the conductances in the two models as well. The smaller A-type K conductance in the CA2 model could be a major factor contributing to differences in D spike propagation between the models of the two cell types. To test this idea rigorously, the authors should put the CA1 conductances into the CA2 cell, and vice versa. If morphology is truly the main difference, as the authors claim, it should still be true that the chimeric model shows better D spike propagation in CA2 than in CA1, despite the reversal of the conductances in the two models.1) Modeling. The current work leaves little doubt that distal PP stimulation results in dendritic Na2) The authors do not show convincingly that the morphological differences between CA1 and CA2 that are apparent in the model are truly representative. To make this conclusion, quantitative data on the morphological properties of the dendritic trees of these two cell types are needed, as opposed to the anecdotal data shown here (one reconstructed neuron of each type). These data, on their own, could bolster the case that morphological differences are real and therefore could account for the differences in D spike propagation. Using modeling to explore more deeply how quantitative morphological differences contribute to D spike initiation/propagation would also strengthen the paper.+ spikes to be generated in these processes, resulting in a large summated response at the primary dendrite which can then effectively propagate to the soma and initiate action potential firing. However, a more detailed analysis is needed. Specifically, in the simulations, are D spikes generated more readily in the distal dendrites of CA2 compared to CA1, or do they propagate better? One possibility is that the major branch point of the main apical dendrite is closer to the soma in CA2. If this is where D spikes fail, they could be larger in CA2 simply because that location is closer to the soma. Is that what's happening or is there some other explanation?3) The authors' simulations indicate that distributed synaptic input across multiple secondary branches of the CA2 dendrite  allows for independent Na4) The paragraph discussing the previous work in CA1 neurons  misses a+ spikes are a requirement for PP-mediated somatic spiking as, following dendrite-targeted TTX application, PP stimulation no longer evoked somatic action potentials. In a few places, the authors claim that D spikes are “necessary” for firing in response to stimulation of the PP. However, this seems like a rather sweeping conclusion given the limited conditions used to stimulate PP inputs. Other conditions not tested in the experiments (e.g. in vivo) could lead to PP-mediated action potentials even in the absence of D spikes. This is easily corrected with some wording changes. For example, rather than saying, “Thus, cortically-driven dendritic Na+ spikes are necessary for AP output in CA2 PNs to either single or bursts of PP input.”, they should say, “Thus, cortically-driven dendritic Na+ spikes were necessary for AP output in CA2 PNs to either single or bursts of PP input.” The difference is subtle. The latter sentence refers only to the experiments the authors did, while the former suggests that it would always be true. Additional discussion could also clarify this point.5) The authors imply that dendritic Na 1) Modeling. The current work leaves little doubt that distal PP stimulation results in dendritic Na+spiking that can facilitate somatic action potential initiation including in physiological-like conditions (even in the presence of GABA-mediated inhibition). However, the authors' conjecture that morphology alone seems sufficient to explain efficiently propagation of dendritic spikes in CA2 PCs is rather simplistic and relatively untested. Although the authors attribute the differences between CA1 and CA2 cells to morphology, they actually have differences in the conductances in the two models as well. The smaller A-type K conductance in the CA2 model could be a major factor contributing to differences in D spike propagation between the models of the two cell types. To test this idea rigorously, the authors should put the CA1 conductances into the CA2 cell, and vice versa. If morphology is truly the main difference, as the authors claim, it should still be true that the chimeric model shows better D spike propagation in CA2 than in CA1, despite the reversal of the conductances in the two models.+ and delayed rectifier K+ conductances – in the CA2 and CA1 PNs. We present these new results in The reviewers raise an important point. Based on the reviewers’ comment, we asked whether it is possible to explain differences in CA2 versus CA1 excitability without postulating a difference in A-type conductance. We now find that, with slight adjustments in the models’ voltage-gated conductance parameters, we can reproduce our experimental results based solely on morphological differences, using an identical value of A-type conductance – as well as identical Na2) The authors do not show convincingly that the morphological differences between CA1 and CA2 that are apparent in the model are truly representative. To make this conclusion, quantitative data on the morphological properties of the dendritic trees of these two cell types are needed, as opposed to the anecdotal data shown here (one reconstructed neuron of each type). These data, on their own, could bolster the case that morphological differences are real and therefore could account for the differences in D spike propagation. Using modeling to explore more deeply how quantitative morphological differences contribute to D spike initiation/propagation would also strengthen the paper.We now have included modeling results from two more reconstructed CA1 and CA2 PNs new , and per3) The authors' simulations indicate that distributed synaptic input across multiple secondary branches of the CA2 dendrite  allows for independent Na+spikes to be generated in these processes, resulting in a large summated response at the primary dendrite which can then effectively propagate to the soma and initiate action potential firing. However, a more detailed analysis is needed. Specifically, in the simulations, are D spikes generated more readily in the distal dendrites of CA2 compared to CA1, or do they propagate better? One possibility is that the major branch point of the main apical dendrite is closer to the soma in CA2. If this is where D spikes fail, they could be larger in CA2 simply because that location is closer to the soma. Is that what's happening or is there some other explanation?We appreciate the reviewers raising this issue. Our data suggest that, compared to CA1, the better propagation due to dendritic geometry of CA2, rather than more excitable dendrites, are important for coupling dendritic spikes to AP output. Our experimental dendritic whole cell current-clamp recordings show the current threshold to generate local spikes is comparable between CA1 and CA2 (CA1: 1.6 ± 0.2 nA versus CA2: 1.7 ± 0.2 nA), indicating that the excitability of CA2 dendrites is comparable to CA1. This point is further supported by our models, in which CA1 distal dendrites can readily generate local spikes . To allo4) The paragraph discussing the previous work in CA1 neurons  misses an important point. The fact that only a small minority of CA1 cells exhibit somatically recorded spikelets likely reflects the fact that D spikes fail so far from the soma that a spikelet is not visible in somatic recordings, rather than reflecting the absence of a D spike in response to PP stimulation. The 2005 study used modeling to predict this result. Those authors also predicted that coincident stimulation of the Schaffer collaterals could facilitate forward propagation of D spikes generated in the tuft. They then tested this prediction experimentally and found that it was indeed the case. Thus, there is no need to invoke a difference between rats and mice to explain the lack of spikelet observed in CA1 in the present study. Rather, it is likely the D spikes are also generated in CA1 tuft dendrites, but their failure to propagate reliably results in so much attenuation that the D spikes are completely invisible in the soma. This point should be discussed.We agree with the reviewers. Our experimental and theoretical results confirm that dendritic spikes are generated in distal CA1 dendrites but are severely attenuated at the soma. We now discuss this important point more clearly.5) The authors imply that dendritic Na+ spikes are a requirement for PP-mediated somatic spiking as, following dendrite-targeted TTX application, PP stimulation no longer evoked somatic action potentials. In a few places, the authors claim that D spikes are “necessary” for firing in response to stimulation of the PP. However, this seems like a rather sweeping conclusion given the limited conditions used to stimulate PP inputs. Other conditions not tested in the experiments (e.g. in vivo) could lead to PP-mediated action potentials even in the absence of D spikes. This is easily corrected with some wording changes. For example, rather than saying, “Thus, cortically-driven dendritic Na+spikes are necessary for AP output in CA2 PNs to either single or bursts of PP input.”, they should say, “Thus, cortically-driven dendritic Na+spikes were necessary for AP output in CA2 PNs to either single or bursts of PP input.” The difference is subtle. The latter sentence refers only to the experiments the authors did, while the former suggests that it would always be true. Additional discussion could also clarify this point.We agree that the necessity of dentritic spikes for AP output in CA2 holds true only under our experimental conditions, and thus have clarified this point based on the reviewers’ comment.


Increasing the dyadic fraction of a particular Ca2+ efflux pathway increases the amount of Ca2+ removed by that pathway, with corresponding changes in Ca2+ efflux from the bulk cytoplasm. The magnitude of these effects varies with the proportion of the total Ca2+ removed from the cytoplasm by that pathway. Differences in the response to EGTA and BAPTA, including changes in Ca2+-dependent inactivation of the L-type Ca2+ current, resulted from the buffers acting as slow and fast “shuttles,” respectively, removing Ca2+ from the dyadic space. The data suggest that complex changes in dyadic Ca2+ and cellular Ca2+ cycling occur as a result of changes in the location of Ca2+ removal pathways or the presence of exogenous Ca2+ buffers, although changing the distribution of Ca2+ efflux pathways has relatively small effects on the systolic Ca2+ transient. We have used a previously published computer model of the rat cardiac ventricular myocyte to investigate the effect of changing the distribution of Ca To simulate the addition of EGTA and BAPTA to the intracellular compartments from a patch pipette, the diffusion of Ca2+ and exogenous Ca2+ buffers (with or without bound Ca2+) among the pipette, subsarcolemmal spaces, dyadic spaces, and cytosol was described using ordinary differential equations where the flux of Ca2+ and Ca2+ buffer between adjacent compartments is directly proportional to the concentration difference of Ca2+ or Ca2+ buffer between the compartments and inversely proportional to the time constant of Ca2+ or buffer exchange, as described previously e = 1 mM; [Na+]e and [K+]e≈ 0 ; [Ca2+]p = 0.5 nM; [Na+]i and [K+]i≈ 0 , reflecting their dialysis via the pipette. K+ currents were disabled because they were blocked experimentally using Cs+  in the network SR (NSR) increased by 9.13%, while that in the cytoplasm increased by 45.6% to 275 nM; the peak of the dyadic Ca2+ transient at the t-tubules increased by 9.9%, while the peak of the cytoplasmic Ca2+ transient increased by 30.2%. Action potential duration at 50% repolarization (APD50) increased by 16%, and Ca2+ influx via ICa increased by 11.8%. These data highlight the interaction between the different Ca2+ efflux pathways and their location, showing that changes in the location of a single pathway can alter the activity of the other Ca2+ flux pathways, as well as Ca2+ concentrations in subcellular spaces and the electrophysiology of the cell.We also explored the effect of simultaneously increasing the dyadic fraction of the three Ca2+ and cytoplasmic plus dyadic Ca2+, respectively. Given the effects of changing the distribution of Ca2+ efflux pathways on Ca2+ dynamics described above, we have investigated the effects of these buffers on Ca2+ dynamics in the model .EGTA and BAPTA have been widely used experimentally to buffer bulk cytoplasmic Ca2+ fluxes and concentrations shown in 2+] in the dyadic and cytoplasmic spaces, EGTA decreased the amplitude of the dyadic Ca2+ transient by ~17%, but it almost completely abolished the cytoplasmic Ca2+ transient, decreasing its amplitude by >90%. In contrast, BAPTA decreased the dyadic Ca2+ transient by ~86% and completely inhibited the cytosolic Ca2+ transient. As a result, these buffers had different effects on Ca2+ fluxes.2+ extrusion via NCX during the initial 170 ms of the cycle as a result of the decreased Ca2+ transient in the dyadic and subsarcolemmal spaces. However, subsequent Ca2+ extrusion via NCX, until the end of the cycle, was higher than in the absence of EGTA, as a result of higher [Ca2+] in both spaces  due to Ca2+ release from the buffer. Similar changes were observed for the SL Ca2+ pump and SERCA, although for these extrusion pathways the increase of Ca2+ extrusion during the later phase of the cycle is greater than its decrease during the Ca2+ transient. Similarly, the faster Ca2+ buffer BAPTA decreased Ca2+ extrusion via NCX during the initial phase of the cycle (80 ms). However, subsequent Ca2+ extrusion via NCX was substantially greater in the presence of BAPTA as a result of the higher [Ca2+] in both spaces  due to Ca2+ released from the buffer. A similar change was observed for the SL Ca2+ pump. The different effect of EGTA and BAPTA on ICa reflects their different efficiency to reduce Ca2+ transient in the dyads and thus to suppress Ca2+-induced inactivation of ICa.EGTA decreased Ca2+ transferred by each efflux pathway , Ca2+ concentrations in intracellular spaces, and AP duration. Over the concentration range investigated, Ca2+ extrusion via SERCA is increased by EGTA and decreased by BAPTA, while the opposite holds for extrusion via NCX. The complex relationship between [buffer] and Ca2+ flux via SERCA is due to the dynamics of [Ca2+] changes in the dyads and the cytosol during the two phases described for 2+ flux via SERCA is reduced during the Ca2+ transient, since Ca2+ transient amplitude is reduced by the buffer; following the Ca2+ transient, Ca2+ flux via SERCA is enhanced as Ca2+ is released from the buffer; this results in an increase of [Ca2+]NSR,end (by 16% for EGTA and 143% for BAPTA). 2+ influx via ICa and the associated APD50 slightly , BAPTA at the same concentration affects both profoundly  as a result of increased Ca2+ entry via ICa due to a marked decrease in CDI as a result of the smaller dyadic Ca2+ transient.The concentration dependence of the effects of EGTA and BAPTA is illustrated in 2+ buffering in the dyad and bulk cytoplasm and, thus, on CDI of ICa, as suggested from the experimental data. We, therefore, investigated in more detail whether the model replicates the effect of these buffers on ICa and the mechanism of Ca2+ buffering.These data are consistent with the idea that EGTA and BAPTA have differential effects on Ca2+) and BAPTA (used to buffer cytoplasmic and dyadic Ca2+) on ICa in the model, compared with the experimental data. ICa in the presence of EGTA and BAPTA  and the time required for the current to decay to 0.37 of the peak amplitude during inactivation (below). Corresponding records and data from the model obtained under the same conditions  between the dyadic and adjacent subsarcolemmal spaces are critical to the efficacy of Ca2+ buffers in the dyadic space. Although both buffers inhibited the cytosolic Ca2+ transient . As a consequence, the concentration gradients of free BAPTA and Ca2+-BAPTA and free EGTA and Ca2+-EGTA between the dyadic space and bulk cytosol were substantially different after the onset of depolarisation. In the case of BAPTA, fast onset of large gradients provided large driving forces, causing rapid movement of Ca2+-BAPTA molecules out of the dyadic space and of free BAPTA into the dyadic space, so that BAPTA appeared to act as a fast “shuttle.” The slower rate of Ca2+ binding to EGTA and the consequent development of substantially smaller free EGTA and Ca2+-EGTA gradients between the dyadic space and cytosol reduced the ability of EGTA to affect dyadic [Ca2+] and, thus, Ca2+-induced inactivation of ICa. The corresponding [Ca2+] in the dyads and its effect on CDI of ICa are shown in the other panels of To understand these changes in more detail, we investigated the differences in concentration of Caransient , only BAICa at the t-tubules  and, thus, CDI of ICa.Addition of EGTA or BAPTA to the model cell resulted in changes of mentally . In addiICa at the t-tubular and surface membrane dyads. The effect of both buffers on CDI and, thus, on ICa, CICR, and the Ca2+ transient is greater at the t-tubular dyad than at the surface dyad (The present work also suggests that EGTA and BAPTA have different effects on CDI of ace dyad , becauseICa at the t-tubules results in t-tubular ICa that has the configuration required for an effective trigger for CICR, while the slower inactivating ICa at the surface membrane has the configuration required to load the SR with Ca2+ for subsequent release [Ca at the t-tubules may also mean that they are an important site for Ca2+ autoregulation (see ICa to SR Ca2+ release may result in an effective trigger for CICR and effective regulation of cell Ca2+ balance, controlled by SR Ca2+ release; conversely, extradyadic ICa may inactivate more slowly, thus loading the SR with Ca2+, suggesting that this Ca2+ may access extradyadic SERCA, and would play less of a role in autoregulation, as a result of being less effectively modulated by SR Ca2+ release.It has been suggested that the more marked CDI of  release . The gretion see . Thus, e


AML patients are reported to have impairments of immune cells which contribute to leukemia progression. Tumor-derived exosomes (TEX) have recently emerged as carriers of the molecular and genetic cargo with potent immunosuppressive properties. We showed that plasma of newly-diagnosed AML patients prior to any therapy contained high levels of exosomal proteins relative to those in plasma of normal donors (NC). AML exosomes were enriched in membrane-associated TGF-β1, MICA/MICB and markers of myeloid blasts. We hypothesize that these plasma-derived virus-size (30-100nm) membrane-bound vesicles operating in AML deliver suppressive signals to immune cells and thus may promote leukemia progression.Venous blood (20-50 mL) was obtained from patients newly diagnosed with AML prior to any treatment, from AML patients who achieved CR following initial therapy and from age-matched NC. Exosome fractions were isolated from plasma using mini-size exclusion chromatography (mini-SEC) with Sepharose 2A. Exosome protein levels, numbers and size of exosomes (qNano) and their morphology (TEM) were determined. Exosomes were characterized by Western blots for expression of exosome markers, Tsg101 and CD81, and myeloid cell-surface markers associated with AML, interleukin-3 receptor alpha chain (CD123) and C-type lectin-like molecule-1 (CLL-1), CD44 and CD96. Isolated normal human NK cells were co-incubated with AML exosomes and multiparameter flow cytometry was used to monitor changes in expression levels of NKG2D on NK cells.Exosome fractions isolated from AML patients' plasma at diagnosis (n=16) had higher mean protein content than fractions obtained from NC plasma . Exosomes were isolated from paired AML plasma samples obtained at diagnosis and in CR after chemotherapy (n=13). In the exosome fractions isolated at CR, at the time when leukemic blasts were undetectable in the bone marrow by conventional hematopathological methods, protein levels remained elevated . AML exosomes isolated at CR carried blast markers, CD123, CLL-1, CD44, CD96 and were enriched in the mature form of TGF-β1. Co-incubation of these exosomes with activated normal human NK cells resulted in down-regulation of NKG2D expression with a concomitant reduction of NK-cell cytotoxicity. Ten/13 AML patients in CR whose plasma exosome levels remained elevated eventually relapsed.The exosome fractions in plasma of patients with AML are enriched in blast-derived exosomes carrying leukemia blast markers and biologically-active TGF-β1. The persistently elevated levels of these exosomes in plasma of AML patients in CR contribute to impaired anti-leukemia immune responses and promote leukemia progression.