Tuberous sclerosis complex (TSC) is an autosomal dominant disorder. It is characterized by seizures, mental retardation and hamartomatous lesions, including facial angiofibroma, subependymal giant cell astrocytoma, cardiac rhabdomyoma and renal angiomyolipoma (AML). AMLs can bleed severely in the retroperitoneal space.Herein, we present the case of a TSC patient presenting with recurrent severe episodes of retroperitoneal hemorrhage from AMLs successfully managed by angio-embolization.Transarterial embolization is effective in preventing and controlling hemorrhage in patients with AMLs of the kidney Tuberous sclerosis complex (TSC) or Bourneville's disease is a rare genetic disorder, either sporadic or inherited by an autosomal dominant transmission. Mutations in one of two genes, TSC1 and TSC2, have been identified as causes of TSC. Affected patients develop hamartomatous lesions in different organs, most commonly in the brain, eyes, heart, kidney, skin and lungs. Angiomyolipomas (AML) of the kidney are the most common TSC lesion, occurring in 70 to 80% of adults and older children.[In September 2000, a 48 year-old Caucasian male with sporadic tuberous sclerosis presented to the emergency room with acute right flank pain, large abdominal mass, signs and symptoms of internal bleeding . Abdominal CT scan showed bilateral renal giant angiomyolipomatosis with a honey-comb aspect of the kidneys and a right retroperitoneal haematoma. Moreover, an arterial contrast blush was seen within the right retroperitoneal hemorrhagic infarction Figure . The patThe three major renal manifestations of TSC include AML and less frequently polycystic kidney disease (PKD) and renal cell carcinoma (RCC), arising alone or in combination. Of these, however, only AML are a major criterion for TSC diagnosis because neither the presence of cysts nor RCC is sufficiently specific for TSC. Angiomyolipomas are benign tumors composed of abnormal vessels, immature smooth muscle cells and fat cells. Their incidence in the general population is 1–2%, whereas in patients with TSC the incidence of AML has been estimated at 50–80%. In respect to sporadic AML, patients with TSC significantly have multiple and bilateral AML that tend to grow with time. As AML grow, aneurysms develop inside the mass. Bleeding from ruptured aneurysm increases in frequency when tumors become larger than 4 cm. Giant renal AML in Bourneville's syndrome raise a surgical interest because they are a source of life-threatening hemorrhage. Hemorrhage from AML has been reported among the most common causes of death in patients with TSC. In the pTransarterial embolization is effective in preventing and controlling hemorrhage in patients with AMLs of the kidney. When AMLs are associated to TSC, recurrence of bleeding is not uncommon and redo angio-embolization may be required.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MC and LD carried out the literature research and drafted the manuscript. MP and MC were major contributor to the writing of this case. CV retrieved and processed the images. All authors read and approved the final manuscript.


MLL) rearrangement. By molecular tracking, we were able to show that MLL cleavage preceded gene rearrangement by 3 months and before the clinical diagnosis of t-AML in a patient with haemophagocytic lymphohistiocytosis. This is the first report on the sequential detection of the two biomarkers in treatment-related leukaemogenesis.Treatment-related acute myeloid leukaemia (t-AML) is a serious complication of topoisomerase 2 inhibitor therapy and is characterised by the presence of mixed lineage leukaemia ( MLL) gene is now well documented . In addition to its clinical importance as an adverse outcome of chemotherapy, t-AML provides an opportunity to investigate the timing of the molecular events involved in the pathogenesis of this condition in relation to the administration of topo 2 inhibitor therapy. We have backtracked the molecular events from a t-AML in a child with Epstein–Barr virus (EBV)-related haemophagocytic lymphohistiocytosis (HLH).The relationship between topoisomerase 2 (topo 2) inhibitor therapy and the pathogenesis of treatment-related acute myeloid leukaemia (t-AML) with rearrangements of the mixed lineage leukaemia (cumented . This ty−2) given twice weekly in the first 2 weeks and then weekly for 5 weeks (cumulative etoposide dose: 1.35 g m−2). At 2 months after the start of treatment, the patient was in complete remission, but by the 6th month after HLH diagnosis (4 months after treatment ceased), he presented with jaw swelling and cervical lymphadenopathy. Bone marrow examination revealed 70% infiltration with myeloid blasts, confirmed by cytochemistry and immunophenotyping as monoblastic AML. There was no CNS involvement by leukaemia. Interphase cytogenetics of the bone marrow showed a chromosome 9;11 translocation ). The patient received induction chemotherapy  for his AML according to the MRC 10th AML trial . The DNA was size fractionated by 0.7% agarose gel electrophoresis, and blotted onto nylon membranes. MLL cleavage was detected, after hybridisation overnight at 60°C with the 32P-labelled MLL probe, using real-time autoradiography. Panhandle PCR method  obtained 3 months after HLH diagnosis, 1 month after the last dose of etoposide (cumulative dose: 1.35 g m−2) and 3 months prior to the diagnosis of overt t-AML. Analysis of Southern blots of the same BamH1-digested DNA samples revealed two rearranged MLL fragments , in addition to the 8.3 kb germline MLL fragment, only in the bone marrow obtained at the time of t-AML diagnosis  MLL PCR product was only detected in the same bone marrow sample  of the fusion gene.The occurrence of t-AML involving MLL-GAS7 within 6 weeks of the start of neuroblastoma therapy including doxorubicin. This was some 15.5 months before the appearance of clinically overt t-AML. The result that we obtained suggests that once established, the putative MLL-AF9 leukaemic clone developed much more rapidly into overt AML as compared to the MLL-GAS7 clone. It also suggests that partner sequences in MLL fusion genes could modify the leukaemogenic process.Our results differ from those reported by MLL cleavage and rearrangement have been detected consecutively in a patient developing t-AML. Our finding thus suggests that the two are temporally related, with gene cleavage and DNA processing prior to translocation, as proposed in the work by As far as we are aware, this is the first case of t-AML in which


Apis cerana cerana (Acc), and the European bee Apis mellifera ligustica (Aml). Using video recordings made at an observation hive, we first confirm that Acc and Aml have significantly different dance dialects, even when made to forage in identical environments. When reared in the same colony, these two species are able to communicate with each other: Acc foragers could decode the dances of Aml to successfully locate an indicated food source. We believe that this is the first report of successful symbolic communication between two honeybee species; our study hints at the possibility of social learning between the two honeybee species, and at the existence of a learning component in the honeybee dance language.The honeybee waggle dance, through which foragers advertise the existence and location of a food source to their hive mates, is acknowledged as the only known form of symbolic communication in an invertebrate. However, the suggestion, that different species of honeybee might possess distinct ‘dialects’ of the waggle dance, remains controversial. Furthermore, it remains unclear whether different species of honeybee can learn from and communicate with each other. This study reports experiments using a mixed-species colony that is composed of the Asiatic bee  When foraging honeybees find an attractive food source, they can perform a special communicative behaviour called the dance language, which was first discovered by Karl von Frisch Apis subspecies make the transition between dance types. According to older published distance communication curves Apis florea and Apis mellifera carnica display striking differences in their dialects. Further research has shown that the dance language could be influenced and affected by both genetic factors Apis mellifera carnica and Apis florea do not differ significantly from each other in the waggle phases performed as a function of distance Honeybee colonies achieve fitness through dancing to share food-location information among their nest mates Apis. Although honeybee workers and queens can be reared in any single-species colony, interspecific reciprocal introductions of female larvae between Apis mellifera and Apis cerana have usually failed Apis mellifera workers are accepted into Asian Apis cerana colonies Apis cerana cerana and Apis mellifera ligustica. Using this mixed-species colony, we were able to investigate the communication and learning of the dance language between individuals of different honeybee species.It is therefore necessary to obtain waggle curves of different species made to forage in the same location and at the same time. A mixed-species study on dance language communication is one possible way to investigate this issue. In general, individual honeybees from different species cannot be put together in one colony, because they have their own special odour, and are likely to attack and kill each other quickly Apis cerana cerana (Acc) queen, Acc workers and Apis mellifera ligustica (Aml) workers, and two other mixed colonies consisting of an Aml queen, Aml workers and Acc workers. In the former colonies, the workers cohabited well for more than 20 days, while in the latter colonies, the Acc workers were killed and cleaned up by Aml workers after 2–3 days. Thus, we were only able to use the former mixed-species colony to carry out our experiments . Under normal conditions, i.e. in single-species colonies, Acc foragers consistently had a much greater waggle duration for a given distance than did Aml foragers, as has been previously reported Acc in the single-species colony was significantly steeper than that of Aml bees in the single-species colony  between Acc and Aml in the mixed-species colony  Acc colony 5 meters away from the observation hive position. We monitored the entrances of both colonies when the foragers came back from the feeder. We did not find a single Acc forager from the feeder at this control colony, indicating that the Acc bees at the feeder had been recruited, and had not merely found it through random searching.In such a hive, we found that both Acc workers while they were following Aml dances, as the observation hive was equipped with a movable glass cover. More than 20 followers were marked for each feeder position. An average of 19.5% , and the frequency of recruited Acc at 500 m was greater than that at 400 m  and 600 m . Comparing visit frequencies at 400 m and 600 m, more bees visited the 400 m feeder than the 600 m feeder, but the difference was not significant . Aml foragers recruited by Aml dancers. We can see that the results are similar to those presented in 1,24 = F0.0402, p = 0.8509, ANOVA for Two-stage Nested Design). Once again, the visit frequency of recruited Aml was significant different at 400 m, 500 m and 600 m , and the frequency of recruited Aml at 500 m was greater than that at 400 m  and 600 m . Comparing visit frequencies at 400 m and 600 m, more bees visited the 400 m feeder than the 600 m feeder, but the difference was not significant . Comparison of Acc foragers and Aml foragers recruited by Aml shows that Aml dancers can recruit slightly more Aml foragers to the 500 m feeder than Acc foragers, but there was no significant difference .To assess the accuracy of Acc was also significantly different among 400 m, 500 m and 600 m as shown in 2,9 = F40.79, p = 0.0001, One-way ANOVA). The frequency of recruited Acc at 500 m was greater than that at 400 m  and 600 m , but the visit frequency of recruited Acc at 400 m was not significantly greater than that at 600 m . Aml foragers recruited by Aml dancers to the three feeders. The visit frequency of recruited Aml was significantly different at 400 m, 500 m and 600 m , and the frequency of recruited Aml at 500 m was greater than that at 400 m  and 600 m , but the visit frequency of recruited Aml at 400 m was not significantly greater than that at 600 m . The visit frequency of Aml foragers recruited by Aml dancers was similar to that of Acc foragers .In the second experiment, three identical feeder-stations were located along the Da-Mei canal, in the north-east direction from the hive, at 400 m, 500 m and 600 m respectively. The methodology was similar to the first experiment, and all the bees used in the previous experiment were captured at the start. The results of the second experiment confirmed the results of the first experiment. The visit frequency of recruited Acc dancers were now made to recruit Acc and Aml foragers to a rewarded feeder flanked by two dummy feeders . However, individuals of each species generally displayed a similar likelihood of following dancers from both species  of Apis mellifera in a mixed hive similar to ours commented on the ‘misunderstandings’ that occur when workers of one subspecies follow the dancers of another Apis mellifera may have diverged around 0.67 million years ago Acc bees in our mixed-species colony were almost as successful as the Aml bees in locating a feeder advertised by more experienced Aml dancers, as indicated by our accuracy experiments. The Acc bees following Aml dances were not only recruited to the experimental feeder, but also preferentially chose the one at the correct distance (as indicated by Aml dances), when two unscented dummy feeders were presented at nearer and farther locations. These results highlight the highly conserved nature of not only the dance itself, but also the mechanisms by which the dance is interpreted by follower bees.An early study on dance communication between two subspecies of intraspecific recruitment is always greater than that of interspecific recruitment, although not in a statistically significant manner.In the accuracy experiments, we also noticed that more recruited foragers, regardless of species, visited the nearer feeder at 400 m than the farther one at 600 m. This indicates that some of the recruited foragers perceived the exact directional, but approximate distance, information regarding the rewarded feeder site, and then searched for their final location after exploring the nearer feeder at 400 m. When comparing the visit frequency of foragers recruited by the same species or by a different species, we find that the number of recruited foragers in Acc and Aml dancers might produce active chemicals to attract new Acc and Aml foragers, although new Acc foragers seem more sensitive to the active chemical than do Aml foragers. This is consistent with our observation that Acc honeybees are more likely to follow dances (in absolute terms) than Aml honeybees. Social learning is classically defined as “learning that is influenced by observation of, or interaction with, another animal  or its products” Acc and Aml workers in the mixed-species colony are indicative of just this kind of social learning.Honeybee waggle dancers produce and release behaviourally active chemicals, which attract new foragers to follow dancers, and excite the followers to fly out of the hive Acc bees become more proficient at decoding Aml dances with increasing foraging experience? In other words, do the kinds of ‘misunderstandings’ reported by Boch Acc bees, whose dances contain longer waggle runs, might, after a period of time, learn to perform shorter dances after following only Aml dances, and searching for the advertised resources. The mixed-species colony used in the present study has paved the way for investigating such questions relating to the learning component of the dance language.There has been a considerable amount of research on the sensory basis of the dance language Acc and Aml have paved a new way to study communication and learning between individuals of different species, which will be helpful in understanding the neural mechanisms of the striking dance language of honeybees.We now know that honeybees have a variety of impressive cognitive skills and an amazing learning ability Apis mellifera ligustica (4–6 frames) and Apis cerana cerana (3–4 frames) were kept at the apiary of Huajiachi Campus, Zhejiang University, Hangzhou, China.Six colonies of Apis cerana cerana (Acc) queen, Acc workers and Apis mellifera ligustica (Aml) workers  of Aml and Acc workers. In order to investigate whether Acc foragers in a mixed-species colony could be recruited by Aml dancers to a particular feeder, we designed two experiments. Firstly, eight to ten Aml workers were trained and marked to collect sugar syrup from an artificial feeder placed at different distances  in a south-west direction from the observation hive on the campus of Agricultural School of Zhangzhou. A hive containing only Acc bees was placed five metres away from the mixed hive as a control for the possibility that stray Acc foragers might accidentally find the feeder during their normal foraging activities. When an Acc forager landed at the feeder, it was marked on the thorax with a paint mark; its appearance back at the experimental hive ensured that it came from the mixed-species colony. In this manner, we were able to confirm whether all Acc bees spotted at the feeder were from the mixed-species colony or not. In addition, any marked Acc forager arriving at the feeder a second time was caught and held for the duration of the experiment. This ensured that all the dances advertising the feeder location at the experimental hive were performed by Aml dancers.We set up two mixed honeybee colonies and transported them to the Agricultural School of Zhangzhou, Fujian province, China. The mixed colonies were put into an observation hive consisting of an Aml workers were trained and marked to collect sugar syrup from an artificial feeder placed at different distances  in a south-west direction from the observation hive. We were able to mark Acc workers, who were following Aml dances, on the thorax and abdomen with paint. Then we monitored the feeder, where we caught any newly-recruited Acc foragers, and held them in a bottle for the duration of the experiment.In a second experiment, the observation hive was equipped with a moveable glass cover. Another mixed-species colony was put into this observation hive, and only Acc foragers recruited by Aml dancers and vice versa. We set up an observation hive with a mixed-species colony of an Acc queen, Acc workers and Aml workers on the banks of the Da-Mei-canal in Zhangzhou, Fujian province, China . Only Aml foragers were trained to collect sugar syrup at the 500 m feeder. After three days' training, around 13 Aml foragers had learnt to visit the 500 m station regularly. They were marked with paint, and after a few visits, they started to dance to recruit other foragers at the mixed-species colony. In the tests, the previously used feeders were replaced with fresh unscented feeders. The feeders at the 400 m and 600 m stations were unrewarded. The visit frequency of the newly-recruited foragers was monitored at each feeder-station from 9:00 a.m. to 12:00 a.m., and from 12:30 p.m. to 16:30 p.m.During December 2007 to February 2008 we carried out complementary experiments to assess the accuracy of e, China . In the We monitored the feeders at all three positions, noting the presence of new foragers at the feeders. To be sure that all new foragers came from our experimental hive, any new recruits arriving at the 500 m feeder was marked with a unique colour combination. All such bees were seen to arrive at the observation hive. We only counted the newly recruited foragers. When the marked recruited foragers visited the 500 m feeder, we did not count them again.Acc queen, Acc workers and Aml workers on the banks of the Da-Mei-canal in Zhangzhou, Fujian province, China. The road that ran along one bank of the canal was mostly deserted. The weather was warm enough for foragers to collect syrup, and the natural food sources were limited. The artificial feeders were set in the south direction at 100 m, 200 m, 300 m and 400 m away from the observation hive, respectively. We carried out the experiments from 9:00 a.m. to 12:00 p.m., and from 12:30 p.m. to 16:30 p.m. During these experiments, the weather was sunny (we stopped the experiments during times of heavy cloud cover and rain), and the air temperature ranged between 18–25° Celsius. We made digital video recordings, at the observation hive, of the dances of Acc and Aml foragers who had been marked at the feeder. As controls, we also recorded the dances of foragers from single-species Acc and Aml colonies, who had been trained to feeders at equivalent distances along the same flight path as the mixed-species colony. The waggle durations of at least ten dancers, with five foraging trips per individual from each species, were recorded and analysed using Ulead VideoStudio 9.0 SE DVD software (http://www.ulead.com/vs/) for feeder distances of 100 m, 200 m, 300 m and 400 m.To study the effect of a mixed-species colony on the waggle dance, we set up an observation hive with a mixed-species colony of an Acc and Aml dancers were analysed in the digital video records using Ulead VideoStudio 9.0 SE DVD software (http://www.ulead.com/vs/). Five waggle phases of each individual were measured, and the average was regarded as the dance angle. To ensure the comparability of dances from each species, we recorded and analysed pairs of individual dances occurring within ten minutes of each other for feeder distances of 100 m, 200 m, 300 m and 400 m.The dance angle, corresponding to the angle between the sun's azimuth and the indicated food source outside the hive, is the direction of the waggle run relative to the direction of gravity. Ten pairs of Acc and Aml workers following a dancer in the pure and mixed-species colonies. The number of workers following at least ten dancers from each species was recorded and analysed for feeder distances of 100 m, 200 m, 300 m, 400 m and 500 m.When we analysed the waggle duration data of the dancers, we also counted the number of The dances were evaluated as follows. For each dance, the mean waggle duration was estimated by averaging the waggle durations over all loops. Then, the mean waggle duration for each bee was obtained by averaging the mean waggle durations over all of its dances at that feeder position. Finally, the mean waggle duration of all bees was calculated from the mean waggle durations for the individuals. The standard error of the mean was also calculated and displayed in the graphs.Linear regressions of the data were computed using the GraphPad Prism  statistical analysis package. To compare waggle durations and regression slopes of different data sets, we used the same statistical package, which implemented the slope comparison test described in Sokal and Rohlf (1995) Apis mellifera and Apis cerana in the pure colony and the mixed-species colony, we used Analysis of Variance for Two-stage Nested Design of DPS Software (http://www.chinadps.net/index.htm). To compare the visit frequencies of recruited Acc and Aml at 400 m, 500 m, and 600 m positions, we used a One-way ANOVA and Tukey's test of DPS Software (http://www.chinadps.net/index.htm). We used a t-test with Welch correction to compare the number of Acc and Aml bees following Acc and Aml dancers in the mixed-species colony. To compare the dance angles collected from Acc dancers with those of Aml dancers, we used a pairwise t-test of DPS software To compare waggle durations of Table S1A summary of statistical significant tests to compare waggle duration.(0.05 MB DOC)Click here for additional data file.Figure S1Accuracy of recruitment by Acc dancers. The experiments were carried out on the banks of the Da-Mei-canal in Zhangzhou, Fujian province of China see . Similar(0.97 MB TIF)Click here for additional data file.Movie S1An Acc queen lays an egg while being attended to by lighter-coloured Aml workers.(9.96 MB MPG)Click here for additional data file.Movie S2Apis cerana cerana (Acc) bees (dark abdomens) following the dance of a marked and an unmarked Apis mellifera ligustica (Aml) forager (lighter abdomens) in the mixed-species colony. Both dancers had been trained to an artificial feeder 200 m away from the hive.(9.11 MB MPG)Click here for additional data file.Movie S3Aml and Acc bees following the dance of a marked Acc forager in the mixed-species colony. Like the bees in video S1, this dancer had also been trained to the same 200 m position.(9.02 MB MPG)Click here for additional data file.Movie S4Trophallaxis between a marked Acc worker (right) and an unmarked Aml worker (left).(5.77 MB MPG)Click here for additional data file.


Chemical synapses transmit information via the release of neurotransmitter-filled vesicles from the presynaptic terminal. Using computational modeling, we predict that the limited availability of neurotransmitter resources in combination with the spontaneous release of vesicles limits the maximum degree of enhancement of synaptic transmission. This gives rise to an optimal tuning that depends on the number of active zones. There is strong experimental evidence that astrocytes that enwrap synapses can modulate the probabilities of vesicle release through bidirectional signaling and hence regulate synaptic transmission. For low-fidelity hippocampal synapses, which typically have only one or two active zones, the predicted optimal values lie close to those determined by experimentally measured astrocytic feedback, suggesting that astrocytes optimize synaptic transmission of information. Release of chemical (neurotransmitter)-filled vesicles at neuronal junctions called synapses leads to transmission of information between neurons. In a successful synaptic transmission, a voltage spike  generated by a presynaptic neuron initiates neurotransmitter vesicle release and leads to a small current in the postsynaptic neuron. For many synapses in the central nervous system, the probability that a neurotransmitter vesicle is released in response to an action potential is conspicuously small, raising the question whether transmission failures can in any way prove advantageous. Apart from “induced vesicle release” , vesicles are also released asynchronously . An induced release probability that is too small samples the information poorly, as many of the incoming action potentials do not result in a postsynaptic current response. Maximizing induced release in order to maximize information transmission at a synapse is accompanied by the exceptionable outcome of increased asynchronous release; in addition, both these releases draw from the same neurotransmitter resource pool. A large release rate thus comprising both induced as well as asynchronous release of vesicles can suppress synaptic transmission via either depletion of neurotransmitter resources or desensitization of postsynaptic receptors. In this paper, we propose that the competing dynamics of induced and asynchronous vesicle release gives rise to an optimal release probability. Further, by comparing experimental data of astrocyte-enhanced synaptic transmission with simulations, we argue that synapses enwrapped by astrocytes operate close to our predicted optimum. This optimality is achieved through a closed-loop control circuitry that involves the presynaptic neuron and the synaptic astrocyte. Optimization principles for the nervous system have long been discussed on various levels of organization. Laughlin and Sejnowski In this paper we propose a novel optimization principle for neurotransmitter release rates that operates at a lower level of organization, that of an individual synapse. At a synapse, vesicles of neurotransmitter are released, signaling the arrival of a presynaptic action potential to the postsynaptic neuron. The probabilities of vesicle release, however, are often conspicuously poor, ranging from 0.1 to 0.5 for hippocampal synapses Sustained stimuli can cause activity dependent depression of synaptic transmission via several pathways. Amongst them reduction in vesicular glutamate concentration  and receptor desensitization play a significant role in lowering the amplitude of the postsynaptic response at central glutamatergic synapses 2+, which can be modulated by neuronal activity dependent feedback from synaptic astrocytes. A single astrocyte can potentially oversee around 100 000 synaptic contacts and more than 50% of the excitatory synapses in the hippocampus are associated with astrocytic processes with varying degrees of coverage The key regulator of vesicle release probability is presynaptic Ca3 production and associated IP3 receptor channel mediated opening of Ca2+ stores 2+ due to opening of these stores Although astrocytes are not electrically excitable they can respond to local neuronal activity with an elevation in intracellular calcium concentration 2+ concentration in the presynaptic terminal. Our methodology consists of combining a model for presynaptic vesicle release with a model for the astrocytic calcium dynamics to investigate the role of astrocyte-neuron interaction for selecting the vesicle release rate. To circumvent the limitation of data regarding the specific biophysical mechanism of glutamate release from astrocyte, we directly incorporate the experimentally determined change in neurotransmission as a result of astrocytic Ca2+ response to neuronal firing. This renders our model insensitive to the aforementioned uncertainty.In our model we assume that glutamate released from the astrocytes that binds to presynaptic mGluRs gives rise to a sustained increase in intracellular Ca2+ response to neuronal firing in hippocampal slices was investigated between GABAergic synapses by Kang et al. 2+ and/or IP3 generates the calcium response in the astrocyte. In our model where we argue that the system operates at an optimal level, it becomes essential to consider a closed loop system. The underlying assumption is that Ca2+ sensor protein that allows for the release of a single unit of neurotransmitter follows the same general principles in central synapses 2+ response of the astrocyte to neuronal firing rate in the model compares favorably with Pasti et al. The increase in transmission probabilties as a result of astrocytic Ca2+ concentration.We started with a model for a low-fidelity hippocampal synaptic junction 2+ concentration In addition to stimulated release during action potentials, spontaneous release of vesicles can also occur. The rate of action potential independent release also depends on the presynaptic CaWe reasoned that the aforementioned competition between triggered and spontaneous events should give rise to an optimal value of the domain calcium concentration in the presynaptic terminal. In order to test this hypothesis, we simulated synaptic dynamics that arise in response to a periodic presynaptic train of action potentials with frequencies ranging from 5 Hz to 40 Hz at a fixed (non-evoked) presynaptic background calcium concentration (regardless of its origin). The transmission of the synapse was characterized by the transmission probability for a single spike and/or the power spectrum of the postsynaptic currents events. The power spectrum exhibits a sharp spike at the frequency of the presynaptic action potentials . The decrease in synaptic transmission beyond its optimal value is due to the spontaneous release of vesicles, which becomes more frequent with increasing presynaptic Ca2+ concentration. To demonstrate this point, we simulated the synapse without the spontaneous release events. The results are shown in We observed that synaptic transmission increases with relative Ca3-dependent cascade leading to a rise in intracellular calcium , and is terminated due to slow inactivation of the IP3Rs at high Ca2+concentration. The typical width of the Ca2+ spike therefore is determined by the inactivation rate of the IP3Rs. The presence of a small number of IP3Rs renders the Ca2+ response stochastic 2+-dependent release of additional glutamate from the astrocyte activates neuronal metabotropic receptors and opens presynaptic calcium stores. This mechanism allows feedback from the astrocyte to regulate the vesicle release probability.We extended our model to include the bidirectional signaling between the synapse and its enveloping astrocyte. This was done following the ideas originally discussed by Nadkarni and Jung 2+ concentration of 300 µM  but have a different number of active zones. The results are plotted in one active zone. This changes the parameters. One active zone requires larger domain presynaptic Ca2+ concentration of 430 µM in order to achieve the same baseline transmission probability of P≈0.2. The transmission curves for one active zone and baseline transmission probability of P≈0.2  active zones, but with a variable baseline (i.e. without astrocyte) transmission probability. While for small baseline transmission probabilities the effect of the astrocytic feedback is a significant improvement of the synaptic transmission probability, this is not the case for large baseline transmission probabilities as seen in optimizes synaptic transmission of low-fidelity synapses. We have developed a modeling framework for the bidirectional interaction between neurons and astrocytes at a synaptic junction. This approach expands upon previous work in two important ways. First, we take into account that the content of synaptic vesicles is variable and can be depressed if the synapse runs short of neurotransmitter resource, and second, that the spontaneous release of vesicles uses up neurotransmitter resource and hence interferes with the transmission of the signal entering the presynaptic terminal as an action potential. This leads to a limit of the enhancement of synaptic transmission through astrocytes and hence predicts that there is an optimal astrocytic enhancement. It is striking that the experimentally observed enhancement lies close to our predicted optimum indicating the possibility that the tripartite synapse In our calculations we do not address the issue of how the feedback parameter α itself might be determined. It thus remains an open question whether longer time feedback mechanisms can automatically find the optimal operating point and hence let the synapse optimize itself. There are a variety of such longer-time processes. Astrocytes drive synaptogenesis through secretion of synaptogenic substances 2+ elevations in astrocytes increased transmitter release probability in hippocampal synapses 3 production and which lead to potentiation 2+ which is augmented by Group I signaling regulates asynchronous release. Of course, our model does not consider synapses that only have Group II or Group III receptors.A recent study shows that temporally coincident Caq-coupled receptors in astrocytes to show that selective calcium elevations in astrocytes are not sufficient to affect neuronal ionotropic glutamate receptors activity. However, in the same paper they were able to reproduce their earlier finding 3 uncaging leading to astrocytic calcium elevation causes an increase in spontaneous activity. This enhanced current activity was attributed to enhanced vesicle release due to activation of mGluRs and opening of presynaptic Ca2+ stores and therefore remains consistent with our model.Another recently published study Our calculations demonstrate that the astrocyte-induced enhancement of synaptic transmission depends only weakly on the frequency content of the presynaptic spikes, since the feedback to the neuron reflects integration of neuronal vesicle release over much longer time scales, from seconds to minutes. We also predicted that for a given synaptic baseline transmission probability, synapses with a larger number of active zones are more sensitive to regulation by synaptic astrocytes. While this prediction suggests an advantage for e.g. two versus one active zone, it should not be directly applied to synapses with hundreds of active zones like large calyces and neuromuscular junctions, since their baseline transmission probability is typically very high.2+ concentration of more than 300 µM during an action potential for the Bertram model 2+-influx through voltage-gated Ca2+ channels gives rise to microdomain concentration profiles with potentially large concentrations near the channels and large gradients away from the channels. Thus the required 300 µM must be interpreted as a domain Ca2+ elevation, which, after termination of the action potential would smooth out rapidly to a much smaller overall concentration by diffusion and binding to buffers. Although such a large value is consistent with values in the literature 2+ levels since each active zone can independently release vesicles.It is generally agreed upon that the number of active zones is 1 or 2 in hippocampal synapses. For a low-fidelity synapse 2+, released from stores in the presynaptic terminal,  and/or delayed release from slow buffers if present. Our assumption of a decay time of about one minute is consistent with this overall picture.Our choice of the small decay-constant for Ca2+ flux for transmitter release 2+ alone can account for all release 2+] at Calyx of Held synapse. Indeed, our own attempts to directly derive asynchronous release rates for hippocampal synapses with very few active release zones did not succeed, predicting event rates that were much larger than experimental values . An allosteric model of release was proposed in another attempt to resolve and improve rate predictions 2+ sensors in a Calyx of Held synapse, one for induced release and another for asynchronous release. The two mechanisms operate independently, with the Ca2+ sensor for induced release governing the response during a fast pulse of high calcium concentration and the Ca2+ sensor for asynchronous release governing the response during the sustained accumulation of the Ca2+ between action potentials. This is consistent with our approach which treats these processes separately. In this study, both mechanisms compete for the same neurotransmitter resource and hence asynchronous release could potentially empty the readily releasable pool during sustained elevations of the Ca2+. The two sensor hypothesis is appealing, as it is able to describe a range of dynamics of at the Calyx of Held. We should note, however, that the novel Ca2+ sensor for asynchronous release remains unidentified. Furthermore, it is unclear if this hypothesis would remain valid for the much smaller hippocampal synapses with a single active zone.Asynchronous release of neurotransmitter by synapses is a poorly understood but a well-established phenomenon. Several parallel explanations have been proposed over the years which include requirement of depolarization along with Ca2+. Lowering the threshold too much does not help since a) the vesicle release machinery spends much time in recovery and b) neurotransmitter resource is wasted. Conversely, if the threshold is too high, i.e. the presynaptic Ca2+ levels too low, the transmission probability of information carrying events is small and the performance of the synapse poor.We conclude the paper by pointing to an interesting parallel with an electrical engineering problem, namely the tuning of a detector in the presence of noise. Let us assume that we want to detect a signal that comes embedded in a noisy background by using a threshold detector. After the detector responds to a signal, it has to recover for a certain time interval before it is ready to detect again. In the absence of noise, the design of the detector is quite simple: the smaller the threshold – the better the result. For small noise the same still holds. But for larger noise, there is an optimal threshold While several parallel pathways of signaling exist between neurons and astrocytes 2+, binding to the vesicle release machinery. To describe vesicle release and its dependence on presynaptic Ca2+, we use the model by Bertram, Sherman and Stanley 2+, with 4 different binding rates . Arrival of an action potential before Ca2+ unbinds from these sites enhances vesicle release probability. This mimics short-term synaptic facilitation consistent with decay times of presynaptic Ca due to a single action potential (100 ms) Out of the four binding sites for Ca2+-binding site at each active zone can be bound and unbound and transitions occur according to a Markov process. If binding site j is unbound at time t, the probability Oj that it will be bound within the time interval  is given by cprejk+δt for sufficiently small δt. If binding site j is bound at time t, it will dissociate with the probability jk−δt within the time interval . A vesicle is released from an active zone at time it, when all 4 Ca2+-binding sites are bound and if the presynaptic membrane is depolarized. Once, a vesicle is released from an active zone, the corresponding vesicle release machinery remains inactivated for about 6.3 ms As there are only a few active zones, the release process cannot be described by the average release rate, but rather by a stochastic algorithm. Each Ca2+ concentration it, we determine the amount of released neurotransmitter by using a dynamical model that considers availability and recovery of neurotransmitter resources I is 1−e−a. At the time of vesicle release it, the neurotransmitter content of a vesicle, i.e. ua, is released into the synaptic cleft. The amount of neurotransmitter in the vesicle depends on the available recovered resource, a, as well the utilization parameter u. During a release event the available fraction of resource, a, drops by the amount ua, but recovers this amount within the recovery time of τrec. The fraction of effective neurotransmitter resource in the cleft, e, increases upon the release of a vesicle by the same amount, ua, but decays subsequently through e.g. pickup by transporters within the inactivation time τin. The values of the parameters including u are such to best match experimental traces of activity dependent depression  cytosolic Ca2+ concentration ca and IP3 concentrations p. We have modeled this mechanism previously 3 concentration p obeys the balance equationp and a base-level of IP3, p03 upon the release of neurotransmitter from the presynaptic terminal (described by the Heaviside function Θ); g denotes the glutamate concentration in the synaptic cleft during the action potential, for which we assume a constant value of 200 mM, consistent with previous analysis n and a maximum flux of v. The values of these parameters  from the cytosol to the Endoplasmic Reticulum (ER)2+ concentration) to cytosol (low Ca2+ concentration)3 and ca and hence reflects the requirement of IP3 and Ca2+ as well as positive feedback through calcium-induced calcium release. The variable q describes Ca2+-induced inhibition when cytosolic Ca2+ levels rise too high.For the cytosolic calcium concentration in the astrocyte, we use the relatively simple stochastic version of the Li-Rinzel model qα and qβ, controlling inhibition through the bottom equation in Equation 6, are given by2+ rises too high, the inactivation rate is large, shutting the flux through IP3Rs down. The relatively small number of IP3Rs (here 20) generates stochasticity of Ca2+ release, which is described  with zero mean and correlations according toIPN3 denotes the number of IP3 receptors. All parameters used are listed in The activation and inactivation rates, 2+ response upon the presynaptic release of a vesicle. In previous work In summary, Equations 5–9 describe the astrocytic Ca2+ concentration exceeds a threshold of approximately 200 nM 2+ from presynaptic stores. This is a slow process on the time scale of seconds to minutes, which maybe stretched even longer through binding of Ca2+ to buffers and/or limited Ca2+ extrusion capabilities. In contrast, Ca2+ entering the presynaptic terminal during an action potential through voltage-gated Ca2+ channels leads typically to an increase of Ca2+ on a small domain which decays within about 100 ms after termination of the action potential 2+ concentration prec into the local domain Ca2+ concentration pre,apc entering through voltage gated Ca2+ channels and the Ca2+ concentration pre,stc released from the stores and decaying on a time scale of about a minute, i.e.pre,apc fixed and determined by the release probability P of neurotransmitter-filled vesicle within the duration of one action potential. Although we assume that pre,apc increases instantaneously when the action potential arrives presynaptically, and switches back to basal level instantly at the end of the action potential, a short-term memory of about 100 ms is taken into account by the small unbinding rates of one of the Ca2+ binding sites of the vesicle-release machinery. The second term on the RHS of Equation 10 (source term) describes the linear increase of presynaptic Ca2+ if the astrocytic Ca2+ concentration ac exceeds threshc. The rate constant γ mimics the time-scale of astrocytic potentiation  and used that to set a.We now turn to the feedback part of the model. Glutamate is released from the astrocytes when its cytosolic Cation see  [18]. Th2+ response to neuronal firing is specific for the activation of mGluRs and the downstream effect of IP3 production and Ca2+ release from IP3R mediated stores, we implemented a less specific model for the additional astrocyte-induced presynaptic Ca2+ to accommodate both NMDA mediated 2+ elevation. The only assumption, supported by references, we are making here is that the corresponding kinetics is slow .While our model for the astrocytic Ca2+] therefore remains valid for both presynaptic activation of NMDA as well as mGluRs, independent of the details of the pathway. Consistent with our assumption, a very recent study 2+ being fertile conditions for domination of asynchronous release.Our hypothesis of competition for neurotransmitter resource between induced release and asynchronous due to sustained elevation of presynaptic [CaThe feedback strength α is the most crucial parameter for this study. It is important to keep in mind that we obtained this particular value for α through calibration with the measured value of transmission probability under the (reasonable) assumption that the presynaptic terminal had 2 active zones. We redid all of the parameter estimation with the alternative assumption that the synapse in question had only one active zone. This changes the parameters Click here for additional data file.Figure S2The signal transmission  for a synapse with two active zones is shown as a function of the astrocytic feedback for various presynaptic spiking frequencies. The vertical line denotes the value of α which corresponds to the experimental tripartite synapse.(0.48 MB EPS)Click here for additional data file.


Ca2+-sparks were often observed to spread across the width of these small cells, and could summate to produce prolonged Ca2+-oscillations and contraction. Application of cyclopiazonic acid (20 μM) transiently increased spark frequency and oscillation amplitude, but inhibited both sparks and oscillations within 60 s. Both ryanodine (100 μM) and tetracaine (100 μM) reduced the frequency of sparks and oscillations, while tetracaine also reduced oscillation amplitude. None of these interventions affected spark amplitude. Nifedipine, which blocks store filling independently of any action on L-type Ca2+-channels in these cells, reduced the frequency and amplitude of both sparks and oscillations. Removal of external [Ca2+] (1 mM EGTA) also reduced the frequency of sparks and oscillations but these reductions were slower in onset than those in the presence of tetracaine or cyclopiazonic acid. Cyclopiazonic acid, nifedipine and low external [Ca2+] all reduced SR loading, as indicated by the amplitude of caffeine evoked Ca2+-transients. This study demonstrates for the first time that spontaneous Ca2+-events in small arterioles of the eye result from activation of ryanodine receptors in the SR and suggests that this activation is not tightly coupled to Ca2+-influx. The data also supports a model in which Ca2+-sparks act as building blocks for more prolonged, global Ca2+-signals.Spontaneous Ca Retinae were rapidly removed and arterioles isolated as previously described 2+ Hanks’ solution and centrifuged at 1200 × g for 1 min. The supernatant was aspirated off and the tissue washed again with low Ca2+ medium. The remaining vascular fragments were loaded with Ca2+-indicator by incubation in 1 ml of low Ca2+ Hanks’ solution containing 10 μM Fluo-4AM  at 21 °C for 2 h. The suspension was then diluted with 10 volumes of low Ca2+ medium and vigorously triturated, and 1 ml of this mixture was pipetted into a circular glass-bottomed recording bath on the stage of an inverted microscope (Nikon Eclipse TE300). Microvessel segments between 0.2 and 2.0 mm in length, and with an outer diameter of 20–40 μm, were anchored down with tungsten wire slips and continuously superfused with normal Hanks’ solution at 37 °C. The recording bath was rotated to align the long axis of the arteriole to the x-axis of the microscope. Drug solutions were applied close to the tissue through a 5-way micro-manifold. The solution exchange time was approximately 1 s, as measured using a dye solution.Animal use conformed to the requirements of The Animals (Scientific Procedures) Act (1986) and the principles set out in the Guide for the Care and Use of Laboratory Animals . Male Sprague–Dawley rats (200–300 g) were anaesthetized with CO2.2d-glucose, 5; CaCl2, 2; MgCl2, 1.3; HEPES, 10; pH set to 7.4 with NaOH. The solution used for isolation of microvessels was of the same composition, but contained just 0.1 mM CaCl2. Stock solutions of drugs used were initially prepared as indicated below, and were diluted to the final concentration and applied in pre-warmed bath solution. The drug suppliers and bath concentrations of vehicle (%v/v) were as follows: cyclopiazonic acid ; ryanodine , tetracaine , nifedipine , EGTA .The Hanks’ solution used to perfuse the bath had the following composition (mM): NaCl, 140; KCl, 5; 2.32.3.12+] ([Ca2+]i) were imaged from adjacent smooth muscle cells in segments of intact arterioles. 2D Ca2+-images (−1. Emitted light was filtered (530–560 nm band-pass) and detected using a photomultiplier tube. Data acquisition was controlled with Timecourse software  and images were processed and analyzed with Image J . Fluorescence images were background corrected using an image frame collected without excitation. To allow for variations in dye loading, corrected confocal fluorescence data (F) were normalized using the average resting fluorescence (F0) for each cell, as measured during periods in which there was no increase in fluorescence. Changes in F/F0 were interpreted as changes in [Ca2+]i. Data collection was limited to a maximum of 80 s in linescan mode, after which background fluorescence started to increase due to photodamage. For presentation purposes, image brightness and contrast have been linearly adjusted for maximum clarity, with the same settings being used for all images from a given experiment.Changes in intracellular  measurements were recorded from the arteriolar smooth muscle using the Ca2+-indicator fura-2, as previously described R = F340/F380) were used as a measure of changes in the cytoplasmic Ca2+ concentration.Global  ([Ca2+]i) were routinely observed in myocytes within segments of intact retinal arterioles superfused with physiological solution at 37 °C  averaged 0.46 ± 0.01 (mean ± S.E.M.), with a mean time to peak fluorescence of 38.0 ± 0.5 ms, and a mean value for the full duration at half maximum (FDHM) of 30.6 ± 0.5 ms. Frequency distribution plots showed positive skew in all three parameters , an inhibitor of the Ca2+-ATPase in the SR membrane −1 during the last 15 s of the control period to 0.40 ± 0.07 s−1 during the first 15 s of cyclopiazonic acid treatment , but returned to control levels during the next 15 s (2+-oscillations (P < 0.05), before decreasing back to control levels in the next 15 s.A series of experiments was carried out to determine the mechanisms responsible for the spontaneous Callations A. Summarllations B. Mean a−1 in the final 5 s control period, to 0.01 ± 0.001 s−1 after 50 s exposure to cyclopiazonic acid  to a mean of 12.2 ± 1.9 ms for sparks recorded in the same cells in the period from 30 to 60 s exposure to cyclopiazonic acid . Oscillation frequency was also reduced in these experiments, falling from an average of 1.37 ± 0.09 (15 s)−1 during the last 15 s of the control period, to 0.30 ± 0.09 (15 s)−1 for the period from 45 to 60 s exposure to cyclopiazonic acid .In a separate series of experiments, the effects of cyclopiazonic acid on sparks and oscillations were tested over a longer period of drug exposure. To avoid the effects of photobleaching, scanning was paused after a 20 s control period and recommenced 30 s after the beginning of superfusion with cyclopiazonic acid, allowing recording up to 60 s drug exposure. Spark frequency was decreased by this period of drug application, falling from 0.52 ± 0.11 sparks s2+-sparks and oscillations, so that when the store is depleted following prolonged blockade of the SR Ca2+-ATPase, this activity is inhibited. The next set of experiments was designed to identify the release mechanism responsible.These results indicate that release from the SR plays an important role in generating Ca3.32+-sparks in other vascular smooth muscle preparations are generally agreed to result from the spontaneous opening of RyR gated channels in the SR Ca3.3.12+-release by any pathway secondary to store-depletion 2+-sparks and oscillations, as shown in the linescan and time-plot from 2 adjacent cells in a single arteriole . Oscillation frequency was reduced from 1.37 ± 0.11 (10 s)−1 under control conditions to 0.80 ± 0.11 (10 s)−1 during the first 20 s of ryanodine application (P < 0.001). In those cells in which spontaneous activity persisted in the presence of ryanodine, there were no statistically significant changes in mean amplitude or duration for either sparks or oscillations.A high concentration of ryanodine (100 μM) was used in an attempt to minimise the number of RyRs locked open in a sub-conductance state, as this could produce non-specific reduction in Carteriole A. The av3.3.2−1 for the period from 10 to 20 s after the start of tetracaine superfusion  during control to 1.06 ± 0.16 (10 s−1) in the presence of tetracaine .To further test for RyR-involvement, we applied tetracaine, an inhibitor of RyRs with no known store-depleting action 2+-events seen in the arteriolar smooth muscle result from opening of RyR-gated Ca2+-release channels.Taken together the effects of ryanodine and tetracaine strongly suggest that the spontaneous Ca3.42+ across the plasma membrane could play a role in the generation of spontaneous Ca2+-release events in at least two distinct ways; by triggering the opening of the Ca2+-sensitive Ca2+-release channels in the SR 2+-store itself 2+.Influx of Ca3.4.12+-channels in smooth muscle . Oscillation frequency was reduced from a mean control value of 0.89 ± 0.07 (10 s−1) to 0.48 ± 0.09 (10 s−1) between 20 and 40 s nifedipine exposure (P < 0.01). Average amplitude fell from 2.27 ± 0.34 for 37 oscillations recorded under control conditions to 0.99 ± 0.11 in 37 oscillations recorded from 20 to 40 s after the start of nifedipine application (P < 0.001).Nifedipine, long recognized as an inhibitor of high-voltage activated, L-type Ca3.4.22+-influx across the plasma membrane. In order to test this more directly, a low [Ca2+] bathing solution was used, in which CaCl2 was substituted with MgCl2, and 1 mM EGTA was added as a Ca2+-chelator. Although this produced a reduction in spark frequency and amplitude over the first 20 s of superfusion in the sample record shown, activity recovered somewhat with continued superfusion so that, after 40 s superfusion, spark amplitude and timecourse were similar to control, even though spark frequency remained depressed  to a mean of 0.38 ± 0.10 (10 s−1) during the second 20 s period of superfusion with low [Ca2+]  was applied twice under control conditions in each experiment, and was then applied to the same preparation for a third time after 40 s of the relevant treatment (n = 6, P < 0.001; n = 6, P < 0.05; 2+] solution (1 mM EGTA; n = 6, P < 0.001; 2+ at these times, as caffeine still evoked substantial transients. This suggests that reductions in RyR open probability resulting from reduced SR [Ca2+] may play a more important role in reducing spontaneous activity than the decline in the driving force for Ca2+-release.We have suggested that the reductions in spontaneous activity seen following inhibition of the SR Careatment . Control44.14.1.12+-events seen 2+-sparks in linescan images (e.g., 2+-sparks, which often originated near the plasma membrane, could increase [Ca2+]i across the width of the cell (e.g., 2+]i to the levels necessary for contraction through spark summation.This report confirms our preliminary observations in this preparation and extends them to consider the mechanisms responsible for the spontaneous Caes e.g., . These oll e.g., . They di2+-sparks promote Ca2+-oscillations, then interventions which modulate spark activity should produce parallel changes in the amplitude or frequency of oscillations. This expectation was fulfilled both under conditions in which spark activity was increased (2+] achieved during such localised events can activate Ca2+-sensitive K+-channels 2+-influx by de-activating voltage sensitive Ca2+-channels 2+-activated K+-current 2+-waves and oscillations in retinal arterioles is consistent with observations from smooth muscle cells isolated from mesenteric arteries and portal veins, in which global signals were often observed to originate from foci of high frequency spark activity, dubbed ‘frequent-discharge-sites’ 2+-channels in arteriolar preparations remain to be determined.If Cancreased , and durncreased . The sim4.1.22+] on the luminal side of the channel is decreased 2+] transients were consistently reduced in size after 40 s exposure to cyclopiazonic acid, nifedipine or low [Ca2+] solution (2+]. This implies that there was still a substantial gradient driving Ca2+-release in each case, consistent with the notion that the decline in spontaneous activity seen under each of these conditions reflected failure of channel opening rather than inadequate flux through open channels.The current data supports a model in which sparks within retinal arterioles behave as stochastic, frequency-modulated signalling events, from which more global and prolonged signals can be constructed. The mean amplitude of the sparks was little affected under a variety of conditions in which spark frequency was greatly altered . Depolarsolution , there w4.24.2.12+-uptake into the SR, resulting in store depletion, ultimately reduced spontaneous activity (2+] in response to reduced store uptake. Any such increase could promote activation of Ca2+-sensitive release channels 2+] for a given influx or release. The subsequent reduction in the size and frequency of oscillations, indicates, however, that slowed removal alone cannot explain all the changes seen, and suggests that store depletion is ultimately responsible for the decay in activity, as previously described in arterial muscle treated with thapsigargin Inhibition of Caactivity . The iniactivity , possibl2+] decay due to uptake by the SR Ca2+-ATPase in cardiac microsomes is, however, of the order of 20 s at 37 °C It is instructive to compare the effects of cyclopiazonic acid on sparks and oscillations, since the changes in spark frequency were not associated with any changes in spark amplitude or kinetics during the initial period of increased activity , or its 4.2.22+]i, and yet inhibition was seen both in cells which showed an increase and in those demonstrating a decrease in basal cytoplasmic [Ca2+] (2+] in cardiac and smooth muscle As in other smooth and striated muscles, activation of ryanodine receptors plays a crucial role in spark generation within these arterioles (reviewed in c [Ca2+] A. It see2+-signals seen in these cells. What remains unclear, however, is how the open probability of the release channels is modulated over time, as reflected in the observation of both isolated release events and the summation of events to give more prolonged oscillations at different times at the same site in a given cell (2+] within the SR, or the cytoplasmic concentration of other second messengers, e.g., inositol 1,4,5-trisophosphate or cyclic ADP-ribose (see reviews: Taken together, these findings indicate that RyR-channel activation is a key event in the spontaneous Caven cell A. It canven cell . Modulat4.32+ reduced spontaneous Ca2+-signalling both in the presence of nifedipine and when extracellular Ca2+ was removed (2+-window current under resting conditions, as store operated channels in rat choroidal and retinal arterioles are also nifedipine sensitive 2+-influx in smooth muscle 2+-entry pathways do play an important role in determining basal activity in these vessels, possibly by refilling the SR stores (2+] had no effect on spark amplitude but nifedipine decreased it, even though both treatments produced similar reductions in store content (Inhibition of influx of extracellular Ca removed . NifedipR stores . Identif content . The dro


A retrospective study of rabbits treated against cheyletiellosis was performed to evaluate the efficacy and safety of selamectin or ivermectin in clinical practice.Cheyletiella infestation were collected from two small animal clinics. The rabbits were divided into three groups, based on treatment protocols. Group 1 included 11 rabbits treated with ivermectin injections at 200–476 μg kg-1 subcutaneously 2–3 times, with a mean interval of 11 days. In Group 2, 27 rabbits were treated with a combination of subcutaneous ivermectin injections (range 618–2185 μgkg-1) and oral ivermectin (range 616–2732 μgkg-1) administered by the owners, 3–6 times at 10 days interval. The last group (Group 3) included 15 rabbits treated with selamectin spot-on applications of 6.2–20,0 mgkg-1, 1–3 times with an interval of 2–4 weeks. Follow-up time was 4 months–4.5 years.Medical records from 53 rabbits with microscopically confirmed Rabbits in remission were 9/11 , 14/27  and 12/15  in groups 1, 2 and 3, respectively.All treatment protocols seemed to be sufficiently effective and safe for practice use. Though very high doses were used in Group 2 , the protocol seemed less efficacious compared to ivermectin injections (Group 1) and selamectin spot on (Group 3), respectively, although not statistically significant. Controlled prospective studies including larger groups are needed to further evaluate efficacy of the treatment protocols. Cheyletiella species is reported worldwide. The condition is highly contagious, with three species, C. parasitivorax, C. blakei and C. yasguri normally infesting rabbits, cats and dogs respectively [Cheyletiella mites belong to the order prostigmata and the family Cheyletiellidae. The mites are large (270–540 μm) and very mobile. Adult mites have four pairs of legs with distal combs instead of claws. The mouthpart  has a pair of curved claws characteristic of the mite Cheyletiella. The three species can be separated morphologically only by the shape of a sensory organ in genu 1 on the first pair of legs. The entire lifecycle of approximately 21 days is spent on the host, where they feed on surface epithelia, debris and lymph. Adult Cheyletiella mites can survive a month without feeding in cool temperatures [Parasite infestation with ectively -3. Cheylre large 70–540 μmC. yasguri between dogs and rabbits suggest a low host specificity of the mite [Cheyletiellosis is a zoonosis, causing papular, pruritic dermatitis in humans ,2. Experthe mite . Cheyletthe mite . Lesionsthe mite -3.Cheyletiella infestation is made by demonstration of the mite or eggs in superficial skin scrapings, transparent tape preparations or by flea combing.Diagnosis of Psoroptes cuniculi and Sarcoptes scabei with ivermectin [Cheyletiella mite infestation in rabbits in clinical practice.A variety of antiparasitic treatment protocols have been successfully used for ectoparasitic mite infestation in small animal veterinary practice. In the literature there is evidence of efficacy using fipronil ,4, ivermermectin -13, iverermectin , moxidecermectin , eprinomermectin , selamecermectin ,18 and dermectin . Althougermectin ,20,21, nCheyletiella infestation were verified by a demonstration of Cheyletiella mites through light microscopy examination of skin scrapings, material from flea combing or transparent tape preparations under 4 or 10 × 100 magnifications. Furthermore, the rabbits should have been treated with ivermectin or selamectin and a follow-up should be possible through telephone contact with the owner or through a revisit at the clinic. The following information was collected from the two veterinary settings: descriptions including age, sex, weight, clinical signs, diagnose verification, treatment protocol , number of rabbits in the household, other treatments and evidence of concurrent diseases. A record of overweight was made if the rabbit's weight was more than 20% above the recommended maximum weight of the breed according to the Nordic rabbit standard [Totally 282 medical records of rabbits from two veterinary settings  were reviewed. Of these, 53 rabbits were included in the study. Prerequisites for selection were that clinical signs compatible with standard .®vet. injectable, 10 mg/ml, Merial SAS, Lyon) injections (Group 1), combination of injections and oral administration of ivermectin  (Group 2) and topical selamectin  (Group 3).The rabbits were divided into three treatment groups, ivermectin (Ivomec-1 (range 200–476) and the mean injection interval was 11 days (range 9–21).Group 1 included 11 rabbits, all treated at the Gästrike Animal Clinic, with a mean age of 4.4 years (range 9 months to 7 years) with bodyweights ranging from 1.4 to 4.6 kg. Eight rabbits were male and three female. Most of the rabbits (n = 9) were from single-rabbit households. In the two multi-rabbit households, all in-contact rabbits were treated. The rabbits were treated with ivermectin injections subcutaneously at two (n = 5) or three (n = 6) occasions. The mean dose was 253 μg kg-1, range 618 to 2185) were followed by oral ivermectin, using the injectable formula  twice and administered by the owner. Most rabbits (n = 23) were re-examined 30 days  after the first visit. Depending on the clinical signs at the second visit they were either given no more treatment (n = 2) or continued treatment. Twenty-one rabbits had a second injection, 14 of which had two additional oral ivermectin treatments repeated, with the same doses and interval as initially. Most of the latter were considered to still have clinical signs (n = 8) or to be mite positive after microscopic control (n = 1).Group 2 included 27 rabbits, all treated at the Animal Clinic Roslagstull, with bodyweights ranging from 1.6 to 6.5 kg and with a mean age of 4 years (range 6 months to 9.5 years). Twenty rabbits were intact males while the rest were four females and three castrated males. Most rabbits lived in single-rabbit households (n = 24) in this group as well, and the rest in households with two or more rabbits. All in-contact rabbits were treated in the multi-rabbit households. Treatment consisted of 3–6 ivermectin administrations at a 10 day interval. Initial subcutaneous injections at the first visit (mean dose of 1044 μgkg-1 (range 6.2 to 20.0).Group 3 included 15 rabbits: one treated at the Gästrike Animal Clinic and 14 at the Animal Clinic Roslagstull. The mean age in this group was 2.2 years (range 3 months to 7 years) with bodyweights ranging from 1 to 7.4 kg. Eight rabbits were intact males while the rest were 5 females and 2 castrated males. Two rabbits belonged to multi-rabbit households. All in-contact rabbits were treated. Treatment consisted of administration of selamectin spot on topically at 1–3 occasions. The mean dose of selamectin used was 12.5 mgkgClinical cure was assessed by re-examination or telephone contact with the owner. Treatment results were graded as in remission, relapse or treatment failure at the time of follow-up. Rabbits in remission were defined as having been free from clinical signs at re-examinations during the whole follow-up period. Relapses were defined as being free from clinical signs more than 3.5 months after treatment but showing signs again during the follow-up time. Treatment failures were cases that never cleared from clinical signs during the first 3.5 months or were recorded with relapse during this time. Adverse reactions of treatment were assessed by examination during revisit and by questioning the owner.2 test [Statistical analysis was made by using χ2 test .-1)  and one with Dermocan-shampoo. Side effects  were reported in one rabbit. Although mite-negative at the last visit, this rabbit was found dead a few months after the last treatment. One rabbit experienced pain during injection of ivermectin. The follow-up time in this group ranged from 4 months to 3.5 years (mean time 18 months).Nine rabbits out of 11 were classified as in remission at follow-up, whereas one was graded as treatment failure and one was recorded as relapse  and five were assessed through telephone contact with the owner. Overweight status was recorded in three rabbits and one rabbit had concurrent conjunctivitis. The cage of one rabbit was cleaned mechanically, confirmed by telephone contact with the owner. Relapse, microscopically verified, was seen in seven rabbits within 7 months to 2 years after the last treatment. In this group one rabbit was overweight and one was in general poor condition at relapse. Six rabbits (four confirmed by visit and two by telephone contact) were graded as treatment failures and still showed clinical signs after treatment with 3–6 ivermectin doses (table Twelve rabbits out of 15 were assessed as in remission. Ten of the twelve were assessed through telephone contact and two through re-examination. In this group four rabbits were recorded as having other diseases (three had a stiff back and one had conjunctivitis) and three were overweight. Two rabbits had their cages cleaned mechanically. The only rabbit considered to be a treatment failure was overweight. Two rabbits relapsed within 3.5 and 8 months, respectively, after the last application and were re-treated with the same treatment protocol .This study indicates that all treatment protocols seemed to be sufficiently effective and safe and that cheyletiellosis in rabbits can be successfully treated using ivermectin or selamectin in clinical practice. Neither ivermectin or selamectin is currently approved for use in rabbits in Sweden. In the UK however a topical spot-on preparation containing ivermectin  has just recently been initiated for use in rabbits, guinea pigs and ferrets.Ivermectin and selamectin both belong to the macrocyclic lactones. All macrocyclic lactones have the same primary mode of action, namely to affect the chloride ion channel activity in the nervous system of nematodes and arthropods. Nerotoxicity however has been described in mammals, mainly in cases of ivermectin use in collies (and collie related breeds) and young animals or after overdosing ,7.-1, at a 10–14 day interval during 6–8 weeks [Psoroptes infestation have concluded that ivermectin at the doses of 100–200 μgkg-1 twice, with a 2 week interval, and of 400 μgkg-1 once subcutaneously, are effective in eliminating the mite [The doses of ivermectin recommended for cheyletiellosis in dogs and cats are oral or injectable formulas at 200–300 μgkgthe mite ,13.-1), using 3–4 topical treatments at 2–4 week intervals [-1) spot-on applications at one or two occasions at 2–4 week intervals had a complete recovery [Selamectin has been shown to have persistent efficacy against fleas after topical administration in dogs and cats for at least 28 days after application . Cheyletntervals ,10. Rabbrecovery ,18.Cheyletiella mites after treatment cannot be excluded, as the response to treatment was assessed as clinical cure and not parasitical cure, except in a few cases. Treatment results were therefore graded as in remission only if no relapse was seen during the entire follow-up time and as relapses if the rabbit experienced recurrence of clinical signs during the follow-up time.The results from this field study may have been affected by uncontrolled events and factors. For example the chance that the treated rabbits in this study were asymptomatic carriers of There may be several explanations as to why the combination of injection and oral administration of ivermectin (Group 2) has a tendency to be less effective, independent of treatment length. It is possible for example that oral bioavailability of ivermectin is not high enough in rabbits, explaining the higher number of rabbits experiencing relapse or treatment failure. Lower efficacy and shorter duration of action from orally administered ivermectin have been documented, for instance in cats where most of the drug is eliminated from plasma at 5 days . The dosCheyletiella may be a source of re-infestation. Most owners were urged to clean the cages, although this cleaning could only be verified in a few cases (8/53). Five of the rabbits in Group 1 were also concurrently bathed. These treatments were made both in the treatment failure groups and in the remission group. Other diseases could possibly also influence the incidence of relapse or lack of treatment success. In 13 of the total 53 rabbits concurrent health issues were recorded and 12 were overweight. Rabbits with concurrent disease or overweight however were seen in both treatment failure and remission groups. Time to relapse varied from a few weeks to several years. Five of the rabbits with relapse were living in single-rabbit households. The mean follow-up time in Group 3 was shorter (8.2 months) compared to the two ivermectin groups (13.7–18.0 months). A study with a longer follow-up time in all groups with clinical and microscopically examination at revisit is needed to further validate the results of this study. Such a study should also include a control group. The outcome of larger groups in such a study might result in statistical significance between the different treatment groups.Why some rabbits still experienced clinical signs or relapsed even when living in single-rabbit households remains unanswered. Insufficient cleaning of the environment and in-contact dogs and cats not treated against -1, which is many times the dosage recommended by the manufacturer for treating ruminants and horses. In ruminants the toxicity dosage (4 mg kg-1) is 20 times the recommended dosage, given as injections, and in horses the toxicity dosage  is 10 times the recommended dosage. Teratogenic studies in rabbits performed by the manufacturers concluded that ivermectin orally administrated daily to pregnant rabbits at the dose 6 mg kg-1 resulted in signs of toxicity after 7 days.Possible side effects were only recorded in two cases, both in Group 1, the group receiving ivermectin injections. One rabbit experienced pain after the ivermectin injection and one developed ataxia at the second treatment. It could not be excluded that the symptoms were correlated to the ivermectin injection. The rabbit experiencing a short episode of ataxia at the second injection died a few months after the last treatment, without having had any relapses of neurological signs. No autopsy was performed. The almost total absence of neurotoxicity is remarkable, since the doses used in Group 2  were very high, up to 2732 μgkgResults of this retrospective study suggest that both ivermectin and selamectin are effective and safe for clearance of clinical signs of cheyletiellosis in rabbits. In the group including oral administration of ivermectin (Group 2) a tendency towards a lower efficacy was registered, as compared to Groups 1 (ivermectin injections) and 3 (selamectin spot-on), although not statistically significant. Controlled prospective studies including larger groups are needed to further evaluate efficacy of the treatment protocols.The author(s) declare that they have no competing interests.MM collected and analyzed the data and drafted the manuscript. KB participated in planning the study and helped to draft the manuscript. All authors read and approved the final manuscript.


Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca2+ properties.Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from 2+ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca2+ decay rate. Stress signaling and Ca2+ regulatory proteins were assessed using Western blot analysis.Murine cardiomyocyte contractile function and intracellular Ca2+ properties , along with prolonged duration of contraction and intracellular Ca2+ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca2+ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca2+ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca2+ through a NADPH oxidase-dependent mechanism.Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca Bacillus anthracis (B. anthracis), a spore-forming, Gram-positive bacterium, has been widely known as the causative agent of anthrax B. anthracis spores by oral, cutaneous or pulmonary routes often triggers infection resulting in a high mortality rate associated with severe hypotension B. anthracis infection B. anthracis vegetative bacteria secrete 3 proteins namely protective antigen (PA), lethal factor (LF) and edema factor (EF), forming anthrax lethal toxin (PA and LF) and anthrax edema toxin (PA and EF) 2-terminal of mitogen-activated protein kinase kinases (MEKs) resulting in inactivation of the kinases. EF is a calmodulin-dependent adenylyl cyclase to promote intracellular cAMP accumulation and the associated cellular responses B. anthracis infection by prophylactic treatment with antibodies or vaccines to anthrax toxins 2+ properties (both in vitro and in vivo) as well as the underlying mechanism of action in isolated murine cardiomyocytes. Given the notion that proteins affording protection against ROS may play an important role in cell survival to lethal toxin infection as evidenced by proteomic studies 2+ regulatory proteins were also evaluated in an effort to better understand the mechanism of action behind lethal toxin-induced cardiomyocyte mechanical and intracellular Ca2+ handling responses, if any.Recombinant PA, LF and EF were produced and purified as previously described ad libitum. For acute lethal toxin challenge in vivo, mice were injected intraperitoneally with anthrax lethal toxin  and were sacrificed under anesthesia  at 2, 4 and 18 hrs after lethal toxin injection. The dosages of lethal toxin injection were chosen based on earlier report of overt myocardial dysfunction in vitro lethal toxin exposure, freshly isolated murine cardiomyocytes were incubated with lethal toxin (0.05 – 50 nM) at 37°C for 2 hrs with or without preincubation of the NADPH oxidase inhibitor apocynin (100 nM).All animal procedures described here were in accordance with humane animal care standards outlined in the NIH Guide for the Care and Use of Experimental and were approved the University of Wyoming Animal Care and Use Committee (#A-3216-01). In brief, adult male C57BL/6J mice (5–6 months of age) were kept in our institutional animal facility under well-controlled conditions of temperature (22±2°C), humidity (55±5%) and a 12 h/12 h light-dark cycle with access to food and water 2/95% O2) Krebs-Henseleit bicarbonate (KHB) buffer containing (in mM) 118 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 HEPES, and 11.1 glucose. Hearts were then perfused with a Ca2+-free KHB containing Liberase Blendzyme 4  for 20 min. After perfusion, left ventricles were removed and minced to disperse cardiomyocytes in Ca2+-free KHB buffer. Extracellular Ca2+ was added incrementally back to 1.25 mmol/l. Only rod-shaped myocytes with clear edges were selected for mechanical and intracellular Ca2+ transient studies. Cells were used within 6 hrs of isolation Cardiomyocytes were isolated as described previously. In brief, mice were anesthetized using ketamine and xylazine . Hearts were rapidly removed and perfused with oxygenated . The myocyte being studied was displayed on a computer monitor using an IonOptix MyoCam camera. An IonOptix SoftEdge software was used to capture changes in cell length during shortening and relengthening. Cell shortening and relengthening were assessed using the following indices: peak shortening (PS), maximal velocities of cell shortening and relengthening (± dL/dt), time-to-PS (TPS), and time-to-90% relengthening (TR90) Mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system . In brief, cardiomyocytes were placed in a chamber mounted on the stage of an inverted microscope  and superfused at 25°C with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2+ concentration were inferred from the ratio of fura-2 fluorescence intensity at two wavelengths (360/380). Fluorescence decay time was assessed as an indication of intracellular Ca2+ clearing. Single and bi-exponential curve fit was applied to calculate the intracellular Ca2+ decay constant Myocytes were loaded with fura-2/AM (0.5 µM) for 10 min and fluorescence measurements were recorded with a dual-excitation fluorescence photo multiplier system (Ionoptix). Cardiomyocytes were placed on an Olympus IX-70 inverted microscope and imaged through a Fluor 40x oil objective. Cells were exposed to light emitted by a 75 W lamp and passed through either a 360 or a 380 nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the cells at 360 nm for 0.5 s then at 380 nm for the duration of the recording protocol (333 Hz sampling rate). The 360 nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca2− was monitored by changes in fluorescence intensity resulted from intracellular probe oxidation Intracellular O5 cells/ml. MTT was added to each well with a final concentration of 0.5 mg/ml, and the plates were incubated for 2 hrs at 37°C. The formazan crystals in each well were dissolved in dimethyl sulfoxide (150 µl/well). Formazan was quantified spectroscopically at 560 nm using a SpectraMax® 190 spectrophotometer  (MTT) assay is based on transformation of the tetrazolium salt MTT by active mitochondria to an insoluble formazan salt. Lethal toxin-treated cardiomyocytes (with or without the NADPH oxidase inhibitor apocynin) were plated in microtiter plate at a density of 3×10H9c2 cells, a clonal cell line derived from fetal rat hearts, were purchased from American Type Culture Collection . Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin and streptomycin and maintained in 95% air and 5% CO2 at 37°C. Cells were grown to 80% confluence before incubation with anthrax lethal toxin.Adenovirus containing LC3-GFP construct  was propagated using HEK293 cell line. In brief, cells were infected with LC3-GFP adenovirus. Upon plaque formation, infected cells were collected, washed with PBS, resuspended in culture medium and lysed by three cycles of freezing (dry ice)-thawing (37°C). Cell debris was collected by centrifugation and aliquots of supernatant with viral particles were stored at −80°C. Adenovirus was purified using an Adeno-X Maxi purification kit . H9c2 cells were grown to confluence on Lab-Tek chamber slide. Cells were then infected at an MOI of 2 with adenoviruses expressing LC3-GFP fusion protein. Medium was replaced with fresh DMEM after 6 hrs. Twenty four hrs later, cells were visualized for autophagy using fluorescence microscopy.H9c2 cells transfected with GFP-LC3 adenovirus were treated with or without anthrax lethal toxin. The cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Cells were washed with PBS three times. Cover slips were mounted on the slides using Vecta mountTM AQ-aqueous mounting medium . For autophagy assessment, cells were visualized at 40× magnification using a Olympus BX51fluorescence microscope  and the percentage of cells showing numerous GFP-LC3 puncta (>10 dots/cells) were scored as described previously 2+ regulatory proteins sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and phospholamban, the ER stress proteins BIP (GRP78), pan and phospho-eIF2α, the autophagy markers Beclin-1 and LC3B were assessed. In brief, left ventricular tissue was sonicated in a lysis buffer containing (in mmol/l): Tris 10, NaCl 150, EDTA 5, 1% Triton X-100 and protease inhibitor cocktail followed by centrifugation at 12,000×g for 10 min. Equal amount (30 µg) protein and prestained molecular weight marker were separated using the 7%–12% SDS-polyacrylamide gels in a minigel apparatus , and were then transferred to nitrocellulose membranes . Membranes were blocked for 1 hr in 5% nonfat milk before being rinsed in TBS-T. The membranes were incubated overnight at 4°C with anti-MEK1/2 , anti-phospho-MEK1/2 , anti-ERK , anti-phospho-ERK , anti-JNK , anti-phospho-JNK , anti-p38 MAP kinase , anti-phospho-p38 MAP kinase , anti-eIF2α , anti-phospho-eIF2α , anti-BIP , anti-Beclin-1 , anti-LC3B , anti-Bcl-xL , anti-Bcl-2 , anti-Bax , anti-Bad , anti-cytochrome-C  and anti-caspase-12  antibodies. After incubation with the primary antibodies, blots were incubated with horseradish peroxidase-linked secondary antibodies  for 60 min at room temperature. Immunoreactive bands were detected using the Super Signal West Dura Extended Duration Substrate . The intensity of bands was measured with a scanning densitometer  coupled with a Bio-Rad personal computer analysis software. α-Tubulin was used as the loading control Expression of the stress signaling molecules MEK1/2, ERK, JNK and p38 MAP kinase, the intracellular CaData were presented as MEAN ± SEM. Statistical significance (p<0.05) for each variable was estimated by analysis of variance (ANOVA).in vitro exposure (2 hrs) of lethal toxin displayed a concentration-dependent (0.05 – 50 nM) prolongation in resting cell length and decrease in peak shortening 90) durations although only at the higher concentrations (90) were due to altered intracellular Ca2+ handling, the influence of lethal toxin on electrically-stimulated intracellular Ca2+ transients was examined. 2+ level (FFI) only at the highest concentration (50 nM) while eliciting an overt inhibition in intracellular Ca2+ rise (ΔFFI) and prolongation in intracellular Ca2+ decay  with a much lower threshold. These data suggest possible involvement of intracellular Ca2+-dependent mechanism(s) in acute lethal toxin exposure-elicited cardiomyocyte contractile inhibitory responses.Acute trations . To detein vivo exposure of lethal toxin  triggered a time-dependent mechanical response in cardiomyocyte shortening. Neither 2 nor 4 hrs of lethal toxin exposure was able to elicit any notable effect on resting cell length, PS, ± dL/dt, TPS and TR90. Interestingly, longer exposure duration (18 hrs) of lethal toxin significantly decreased resting cell length and ± dL/dt, as well as prolonged TPS and TR90 without affecting PS in murine cardiomyocytes  significantly decreased resting intracellular Ca2+ level (FFI), and prolonged intracellular Ca2+ decay  without affecting rise in intracellular Ca2+ (ΔFFI) following electrical stimulus -elicited cardiomyocyte contractile dysfunctions.Acute myocytes . None ofstimulus . Similarin vitro lethal toxin exposure on cardiomyocyte shortening was re-examined in the presence of the NADPH oxidase inhibitor apocynin (100 nM). Apocynin alone had little effect on cell shortening over 2 hrs (data not shown), consistent with our previous report in vitro lethal toxin exposure-induced inhibition in PS and ± dL/dt as well as prolongation of TR90 without affecting lethal toxin-elicited action on resting cell length and TPS . Similarly, in vivo examination depicted that pretreatment of mice with apocynin mitigated in vivo lethal toxin exposure-induced superoxide production  significantly down-regulated and up-regulated the expression of SERCA2a and phospholamban, respectively, the effects of which were partially restored or ablated by apocynin pretreatment  for 18 hrs revealed overtly downregulated MEK1/2, phospho-MAP kinase and phospho-ERK associated with hyperactivation of JNK in murine hearts . Our results revealed that lethal toxin produced subtle although significant upregulation in the expression of the ER stress maker BIP and the autophagy markers Beclin-1 and LC3-II without affecting the phospho-eIF2α-to- eIF2α ratio in murine hears  may be underscored by superoxide production and cell death via a NADPH oxidase-dependent mechanism. These results may suggest a likely role of cardiomyocyte dysfunction in anthrax infection-induced pathophysiological change in the heart.Data from our current study provided evidence for the first time that Although cardiac defects triggered by anthrax toxin seem to depend upon the presence of two toxin components, i.e., lethal toxin (PA+LF) and edema toxin (PA+EF) 2+ transient, suggesting a likely role of intracellular Ca2+ homeostasis in lethal toxin-induced inhibition of cardiomyocyte contraction. Our findings of depressed PS and prolonged TR90 following lethal toxin exposure are in line with the reduced intracellular Ca2+ rise (ΔFFI) and slowed intracellular Ca2+ decay or clearing. In general, there is a general agreement in the mechanical and intracellular Ca2+ parameters measured between the in vitro and in vivo lethal toxin studies. The subtle disparity in peak shortening amplitude and intracellular Ca2+ rise (ΔFFI) between in vitro and in vivo lethal toxin exposure may be due to the seemingly discrepant effect of lethal toxin on resting cell length (elongation in vitro and shortening in vivo) as peak shortening amplitude is normalized to resting cell length. The precise mechanism behind lethal toxin-associated disparity in resting cell length is unknown at this point although difference between in vitro and in vivo experimental settings may play a role. In our hand, in vivo lethal toxin exposure failed to elicit any obvious cardiomyocyte mechanical and intracellular Ca2+ handling changes until 18 hrs following treatment. The time-dependent response of in vivo lethal toxin exposure is somewhat supported by our earlier report in vitro data revealed that cellular machineries responsible for cardiac relaxation such as SERCA and phospholamban were downregulated and upregulated, respectively, in response to lethal toxin exposure. These changes are in line with prolonged cardiac relaxation and delayed intracellular Ca2+ decay in response to lethal toxin exposure. SERCA is considered as the single most important machinery to remove Ca2+ from cytosolic space  for cardiac relaxation to occur in vitro lethal toxin exposure, favoring a likely role of SERCA/phospholamban in lethal toxin-induced intracellular Ca2+ dysregulation. Somewhat surprisingly, prolonged in vivo exposure (18 hrs) failed to significantly affect expression of SERCA and phospholamban, suggesting presence of compensatory mechanism or time-dependence in lethal toxin-induced response on Ca2+ regulatory proteins. Furthermore, although our in vitro data do not favor a major role of stress signaling MEK1/2, p38 MAP kinase, JNK and ERK in lethal toxin-elicited cardiomyocyte response in vitro, the fact that these stress signaling molecules were significantly down-regulated  or upregulated (JNK) in the in vivo setting of lethal toxin exposure suggest a time-dependent response for stress signaling activation. Additional studies using myocardial tissues from anthrax lethal toxin-treated animals at various time points are in need to better elucidate these kinase activity as well as the link between various stress signaling activation and cardiomyocyte mechanical alterations under anthrax lethal toxin infection.Several mechanisms may be postulated for lethal toxin exposure-induced cardiomyocyte contractile dysfunction. Our results showed that lethal toxin significantly inhibits cardiomyocyte contractile function as well as intracellular Ca2+ mishandling, intracellular superoxide generation, cell death as well as change in Ca2+ regulatory proteins SERCA2a and phospholamban. These data indicate a key role of NADPH oxidase in lethal toxin-induced cardiomyocyte dysfunction. NADPH oxidase has emerged as a key source of reactive oxygen species with diverse cellular functions including anti-microbial defense, inflammation and redox signaling. NADPH oxidase is expressed in the cardiovascular system and is involved in physiological and pathological processes such as the regulation of cardiac contractility, vascular tone, cell growth, migration, proliferation, hypertrophy, apoptosis and matrix deposition phox and p22phox) and the others are in the cytosol  phox and p67phox translocation and superoxide formation through blockade of sulfhydryl groups thus interrupting the NADPH oxidase enzyme assembly phox and p67phox protein may occur within a short time frame as short as 15 min Perhaps the most novel finding from our study was that NADPH oxidase inhibition with apocynin mitigated lethal toxin-induced cardiomyocyte contractile dysfunction, intracellular Ca2+ mishandling, possibly through NADPH oxidase-mediated superoxide production and cell death. These findings not only reveal a novel role of cardiomyocyte function in anthrax lethal toxin-induced cardiovascular anomalies but also provide new signaling insights for lethal toxin-induced cell injury. The precise mechanism behind anthrax lethal toxin-induced cardiac pathophysiological changes is still unclear. Future studies should focus on its action on cardiac excitation-contraction coupling and membrane ion channels. These approaches will be essential to further our understanding of the pathophysiology and therapeutic strategy of anthrax infection-induced collapse in cardiovascular function.In conclusion, our study demonstrates that lethal toxin triggers cardiomyocyte contractile dysfunction and intracellular CaFigure S1Effect of in vivo lethal toxin (LeTx) exposure on the expression of Bcl-xL, Bcl-2, Bax, Bad, cytochrome C and caspase-12 in murine hearts. Adult male C57BL/6J mice were treated with LeTx  for 18 hrs. A: Representative gel blots of Bcl-xL, Bcl-2, Bax, Bad, cytochrome C, caspase-12 and α-tubulin (loading control) using specific antibodies; B: Bcl-xL; C: Bcl-2; D: Bax; E: Bad; F: Cytochrome C; and G: Caspase-12. Mean ± SEM, n = 4 –5 mice, * p<0.05 vs. C57BL/6J group without LeTX treatment.(0.20 MB DOC)Click here for additional data file.