Comparing critical speed modelling approaches and exploring relationships with match-play variables in elite male youth soccer players.

Background: A novel bi-exponential method has emerged to estimate critical speed (CS) and D-prime (D') from a 3-min all-out test (3MT). Objectives: To compare CS analysis methods to determine whether parameter estimations were interchangeable. Reference values and relationships with key soccer match-play variables were explored. Methods: Thirteen elite male youth (14-15 years old) players completed a 30 m shuttle run 3MT to estimate CS, D', rate of speed decline time constant, maximal speed (S max), time to S max (t max), and fatigue index (FI), using the traditional method and bi-exponential model on average (Bi-ExpAverage) and max speed settings (Bi-ExpMax-Speed). High-speed running (HSR) and sprinting distances and counts, and the number of accelerations were collected from two matches. Magnitude-based inferences (p < 0.05) with smallest worthwhile change of 0.2 effect sizes were used to analyse differences. Pearson's and Spearman's correlation coefficients were used to measure associations between CS model variables and match-play parameters. Results: There were significant differences between the traditional method and both bi-exponential models for CS and D', as well as between the bi-exponential models for all variables except t max. Using the Bi-ExpAverage model, strong correlations (r = 0.70-0.73; p < 0.05) were observed for D' and FI with the number of standardised and individualised HSRs, respectively. With the Bi-ExpMax-Speed model, there were strong correlations (r/ρ = 0.64-0.68; p < 0.05) between D' and the number of standardised HSRs and sprints, and the number of individualised sprints. Conclusion: There is a lack of interchangeability between analysis methods. It appears that D' and FI from the bi-exponential models could be associated with high-intensity actions in soccer match-play.

Evaluation of Mortality in Atrial Fibrillation: Clinical Outcomes in Digital Electrocardiography (CODE) Study.

Aims: Atrial fibrillation (AF) is a public health problem and its prevalence is increasing worldwide. Electronic cohorts, with large electrocardiogram (ECG) databases linked to mortality data, can be useful in determining prognostic value of ECG abnormalities. Our aim is to evaluate the risk of mortality in patients with AF from Brazil. Methods: This observational retrospective study of primary care patients was developed with the digital ECG database from the Telehealth Network of Minas Gerais, Brazil. ECGs performed from 2010 to 2017 were interpreted by cardiologists and the University of Glasgow automated analysis software. An electronic cohort was obtained linking data from ECG exams and those from a national mortality information system, using standard probabilistic linkage methods. We considered only the first ECG of each patient. Patients under 16 years were excluded. Hazard ratios (HR) for mortality were adjusted for demographic and self-reported clinical factors and estimated with Cox regression. Results: From a dataset of 1,773,689 patients, 1,558,421 were included, mean age 51.6 years; 40.2% male. There were 3.34% deaths from all causes in 3.68 years of median follow up. The prevalence of AF was 1.33%. AF was an independent risk factor for all-cause mortality (HR 2.10, 95%CI 2.03-2.17) and cardiovascular mortality (HR 2.06, 95%CI 1.86-2.29). Females with AF had a higher risk of overall and cardiovascular mortality compared with males (p < 0.001). Conclusions: AF was a strong predictor of cardiovascular and all-cause mortality in a primary care population, with increased risk in women. Condensed abstract: To assess risk of mortality in AF patients, an electronic cohort was obtained linking data from ECG exams of Brazilian primary care patients and a national mortality information system. From 1,558,421 patients, AF (prevalence 1.33%) carried a higher risk of overall and cardiovascular mortality, with increased risk in women. What's New: This is the first study with a large Brazilian electronic cohort to evaluate the risk of mortality linked to AF in primary care patients.AF patients from a Brazilian primary care population had a higher risk of death for all causes (HR 2.10, 95%CI 2.03-2.17) and cardiovascular mortality (HR 2.06, 95%CI 1.86-2.29).Female patients with AF had an increased risk of overall and cardiovascular mortality compared with male patients (p < 0.001).

Author Correction: Automatic diagnosis of the 12-lead ECG using a deep neural network.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Automatic diagnosis of the 12-lead ECG using a deep neural network.

The role of automatic electrocardiogram (ECG) analysis in clinical practice is limited by the accuracy of existing models. Deep Neural Networks (DNNs) are models composed of stacked transformations that learn tasks by examples. This technology has recently achieved striking success in a variety of task and there are great expectations on how it might improve clinical practice. Here we present a DNN model trained in a dataset with more than 2 million labeled exams analyzed by the Telehealth Network of Minas Gerais and collected under the scope of the CODE (Clinical Outcomes in Digital Electrocardiology) study. The DNN outperform cardiology resident medical doctors in recognizing 6 types of abnormalities in 12-lead ECG recordings, with F1 scores above 80% and specificity over 99%. These results indicate ECG analysis based on DNNs, previously studied in a single-lead setup, generalizes well to 12-lead exams, taking the technology closer to the standard clinical practice.

Evaluation of mortality in bundle branch block patients from an electronic cohort: Clinical Outcomes in Digital Electrocardiography (CODE) study.

BACKGROUND: Left bundle branch block is recognized as a marker of higher risk of death, but the prognostic value of the right bundle branch block in the general population is still controversial. Our aim is to evaluate the risk of overall and cardiovascular mortality in patients with right (RBBB) and left bundle branch block (LBBB) in a large electronic cohort of Brazilian patients. METHODS: This observational retrospective study was developed with the database of digital ECGs from Telehealth Network of Minas Gerais, Brazil (TNMG). All ECGs performed from 2010 to 2017 in primary care patients over 16 years old were assessed. The electronic cohort was obtained by linking data from ECG exams (name, sex, date of birth, city of residence) and those from national mortality information system, using standard probabilistic linkage methods (FRIL: Fine-grained record linkage software, v.2.1.5, Atlanta, GA). Only the first ECG of each patient was considered. Clinical data were self-reported, and ECGs were interpreted manually by cardiologists and automatically by the Glasgow University Interpreter software. Hazard ratio (HR) for mortality was estimated using Cox regression. RESULTS: From a dataset of 1,773,689 patients, 1,558,421 primary care patients over 16 years old underwent a valid ECG recording during 2010 to 2017. We excluded 17,359 patients that didn't have a valid QRS measure from the Glasgow program and 11,091 patients from the control group that had QRS equal or above 120 ms and were not RBBB or LBBB. Therefore, 1,529,971 were included (median age 52 [Q1:38; Q3:65] years; 40.2% were male). In a mean follow-up of 3.7 years, the overall mortality rate was 3.34%. RBBB was more frequent (2.42%) than LBBB (1.32%). In multivariate analysis, adjusting for sex, age and comorbidities, both patients with RBBB (HR 1.32; CI 95% 1.27-1.37) and LBBB (HR 1.69; CI 95% 1.62-1.76) had higher risk of overall mortality. Women with RBBB had an increased risk of all-cause death compared to men (p < 0.001). Cardiovascular mortality was higher in patients with LBBB (HR 1.77; CI 95% 1.55-2.01), but not for RBBB. CONCLUSIONS: Patients with RBBB and LBBB had higher risk of overall mortality. Women with RBBB had more risk of all-cause death than men. LBBB was associated with higher risk of cardiovascular mortality.

Tele-electrocardiography and bigdata: The CODE (Clinical Outcomes in Digital Electrocardiography) study.

Digital electrocardiographs are now widely available and a large number of digital electrocardiograms (ECGs) have been recorded and stored. The present study describes the development and clinical applications of a large database of such digital ECGs, namely the CODE (Clinical Outcomes in Digital Electrocardiology) study. ECGs obtained by the Telehealth Network of Minas Gerais, Brazil, from 2010 to 17, were organized in a structured database. A hierarchical free-text machine learning algorithm recognized specific ECG diagnoses from cardiologist reports. The Glasgow ECG Analysis Program provided Minnesota Codes and automatic diagnostic statements. The presence of a specific ECG abnormality was considered when both automatic and medical diagnosis were concordant; cases of discordance were decided using heuristisc rules and manual review. The ECG database was linked to the national mortality information system using probabilistic linkage methods. From 2,470,424 ECGs, 1,773,689 patients were identified. After excluding the ECGs with technical problems and patients <16 years-old, 1,558,415 patients were studied. High performance measures were obtained using an end-to-end deep neural network trained to detect 6 types of ECG abnormalities, with F1 scores >80% and specificity >99% in an independent test dataset. We also evaluated the risk of mortality associated with the presence of atrial fibrillation (AF), which showed that AF was a strong predictor of cardiovascular mortality and mortality for all causes, with increased risk in women. In conclusion, a large database that comprises all ECGs performed by a large telehealth network can be useful for further developments in the field of digital electrocardiography, clinical cardiology and cardiovascular epidemiology.

Fifty years my mentor: Harold Atwood.

While readers of Journal of Neurogenetics may be familiar with Harold Atwood's work with Drosophila, most may know little of his previous work on crustacean neuromuscular systems that prepared him to utilise Drosophila neuromuscular junctions. Here, I will give brief overviews of his academic career, one line of his research that persisted throughout his career and his entry to the Drosophila field. This is not a review paper. Finally, I will relate my experiences with Atwood since 1967 as an undergraduate, Postdoctoral Fellow, and Faculty member and finish with some personal anecdotal observations.

Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission.

Oxygen-glucose deprivation (OGD) leads to depression of evoked synaptic transmission, for which the mechanisms remain unclear. We hypothesized that increased presynaptic [Ca2+]i during transient OGD contributes to the depression of evoked field excitatory postsynaptic potentials (fEPSPs). Additionally, we hypothesized that increased buffering of intracellular calcium would shorten electrophysiological recovery after transient ischemia. Mouse hippocampal slices were exposed to 2 to 8 min of OGD. fEPSPs evoked by Schaffer collateral stimulation were recorded in the stratum radiatum, and whole cell current or voltage clamp recordings were performed in CA1 neurons. Transient ischemia led to increased presynaptic [Ca2+]i, (shown by calcium imaging), increased spontaneous miniature EPSP/Cs, and depressed evoked fEPSPs, partially mediated by adenosine. Buffering of intracellular Ca2+ during OGD by membrane-permeant chelators (BAPTA-AM or EGTA-AM) partially prevented fEPSP depression and promoted faster electrophysiological recovery when the OGD challenge was stopped. The blocker of BK channels, charybdotoxin (ChTX), also prevented fEPSP depression, but did not accelerate post-ischemic recovery. These results suggest that OGD leads to elevated presynaptic [Ca2+]i, which reduces evoked transmitter release; this effect can be reversed by increased intracellular Ca2+ buffering which also speeds recovery.

Differential roles of the mevalonate pathway in the development and survival of mouse Purkinje cells in culture.

The cerebellum is an important locus for motor learning and higher cognitive functions, and Purkinje cells constitute a key component of its circuit. Biochemically, significant turnover of cholesterol occurs in Purkinje cells, causing the activation of the mevalonate pathway. The mevalonate pathway has important roles in cell survival and development. In this study, we investigated the outcomes of mevalonate inhibition in immature and mature mouse cerebellar Purkinje cells in culture. Specifically, we found that the inhibition of the mevalonate pathway by mevastatin resulted in cell death, and geranylgeranylpyrophosphate (GGPP) supplementation significantly enhanced neuronal survival. The surviving immature Purkinje cells, however, exhibited dendritic developmental deficits. The morphology of mature cells was not affected. The inhibition of squalene synthase by zaragozic acid caused impaired dendritic development, similar to that seen in the GGPP-rescued Purkinje cells. Our results indicate GGPP is required for cell survival and squalene synthase for the cell development of Purkinje cells. Abnormalities in Purkinje cells are linked to motor-behavioral learning disorders such as cerebellar ataxia. Thus, serious caution should be taken when using drugs that inhibit geranylgeranylation or the squalene-cholesterol branch of the pathway in the developing stage.

A novel extraction protocol to probe the role of cholesterol in synaptic vesicle recycling.

Cholesterol helps to stabilize membrane fluidity and many membrane proteins interact with cholesterol and are functionally clustered in cholesterol rich "rafts." Synaptic vesicle (SV) membranes are enriched in cholesterol in comparison to other organelles. Attempts to study the function of this high cholesterol content have been hampered by the inability to extract cholesterol from SVs in live presynaptic terminals. Here, we describe a method to extract vesicular cholesterol using a temperature-sensitive Drosophila dynamin mutant, shibire-ts1 (shi), to trap SVs on the plasma membrane. Trapped SVs are more accessible to cholesterol extraction by the cholesterol chelator, methyl-ß-cyclodextrin (MßCD). This method can likely be extended to extract other lipids from SVs and could also be used to add lipids. We speculate that this method could be used on mammalian preparations in conjunction with dynamin inhibitors.

SNARE zippering and synaptic strength.

Synapses vary widely in the probability of neurotransmitter release. We tested the hypothesis that the zippered state of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex determines initial release probability. We tested this hypothesis at phasic and tonic synapses which differ by 100-1000-fold in neurotransmitter release probability. We injected, presynaptically, three Clostridial neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by the zippering of the SNARE complex or open to proteolytic attack. Under low stimulation conditions, the catalytic light-chain fragment of botulinum B (BoNT/B-LC) inhibited evoked release at both phasic and tonic synapses and cleaved VAMP; however, neither BoNT/D-LC nor tetanus neurotoxin (TeNT-LC) were effective in these conditions. The susceptibility of VAMP to only BoNT/B-LC indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end to approximately the zero-layer with the C-terminal end exposed under resting state. Therefore, the existence of the same partially zippered state of the trans-SNARE complex at both phasic and tonic synapses indicates that release probability is not determined solely by the zippered state of the trans-SNARE complex at least to the zero-layer.

Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane.

Synaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-ß-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins.

Calcium, calpain, and calcineurin in low-frequency depression of transmitter release.

Low-frequency depression (LFD) of transmitter release occurs at phasic synapses with stimulation at 0.2 Hz in both isolated crayfish (Procambarus clarkii) neuromuscular junction (NMJ) preparations and in intact animals. LFD is regulated by presynaptic activity of the Ca(2+)-dependent phosphatase calcineurin (Silverman-Gavrila and Charlton, 2009). Since the fast Ca(2+) chelator BAPTA-AM inhibits LFD but the slow chelator EGTA-AM does not, the Ca(2+) sensor for LFD may be close to a Ca(2+) source at active zones. Calcineurin can be activated by the Ca(2+)-activated protease calpain, and immunostaining showed that both proteins are present at nerve terminals. Three calpain inhibitors, calpain inhibitor I, MDL-28170, and PD150606, but not the control compound PD145305, inhibit LFD both in the intact animal as shown by electromyograms and by intracellular recordings at neuromuscular junctions. Analysis of mini-EPSPs indicated that these inhibitors had minimal postsynaptic effects. Proteolytic activity in CNS extract, detected by a fluorescent calpain substrate, was modulated by Ca(2+) and calpain inhibitors. Western blot analysis of CNS extract showed that proteolysis of calcineurin to a fragment consistent with the constitutively active form required Ca(2+) and was blocked by calpain inhibitors. Inhibition of LFD by calpain inhibition blocks the reduction in phosphoactin and the depolymerization of tubulin that normally occurs in LFD, probably by blocking the dephosphorylation of cytoskeletal proteins by calcineurin. In contrast, high-frequency depression does not involve protein phosphorylation- or calpain-dependent mechanisms. LFD may involve a specific pathway in which local Ca(2+) signaling activates presynaptic calpain and calcineurin at active zones and causes changes of tubulin cytoskeleton.

Rhodnius prolixus lipophorin: lipid composition and effect of high temperature on physiological role.

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin-layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55 °C. When it was pretreated at 75 °C, maximal inhibition of phospholipid transfer was observed after 3-min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75 °C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55 °C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75 °C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65 °C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85 °C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.

Vesicular sterols are essential for synaptic vesicle cycling.

Synaptic vesicles have a high sterol content, but the importance of vesicular sterols during vesicle recycling is unclear. We used the Drosophila temperature-sensitive dynamin mutant, shibire-ts1, to block endocytosis of recycling synaptic vesicles and to trap them reversibly at the plasma membrane where they were accessible to sterol extraction. Depletion of sterols from trapped vesicles prevented recovery of synaptic transmission after removal of the endocytic block. Measurement of vesicle recycling with synaptopHluorin, FM1-43, and FM4-64 demonstrated impaired membrane retrieval after vesicular sterol depletion. When plasma membrane sterols were extracted before vesicle trapping, no vesicle recycling defects were observed. Ultrastructural analysis indicated accumulation of endosomes and a defect in the formation of synaptic vesicles in synaptic terminals subjected to vesicular sterol depletion. Our results demonstrate the importance of a high vesicular sterol concentration for endocytosis and suggest that vesicular and membrane sterol pools do not readily intermingle during vesicle recycling.

Equilibrative nucleoside transporter 2 regulates associative learning and synaptic function in Drosophila.

Nucleoside transporters are evolutionarily conserved proteins that are essential for normal cellular function. In the present study, we examined the role of equilibrative nucleoside transporter 2 (ent2) in Drosophila. Null mutants of ent2 are lethal during late larval/early pupal stages, indicating that ent2 is essential for normal development. Hypomorphic mutant alleles of ent2, however, are viable and exhibit reduced associative learning. We additionally used RNA interference to knock down ent2 expression in specific regions of the CNS and show that ent2 is required in the alpha/beta lobes of the mushroom bodies and the antennal lobes. To determine whether the observed behavioral defects are attributable to defects in synaptic transmission, we examined transmitter release at the larval neuromuscular junction (NMJ). Excitatory junction potentials were significantly elevated in ent2 mutants, whereas paired-pulse plasticity was reduced. We also observed an increase in stimulus dependent calcium influx in the presynaptic terminal. The defects observed in calcium influx and transmitter release probability at the NMJ were rescued by introducing an adenosine receptor mutant allele (AdoR(1)) into the ent2 mutant background. The results of the present study provide the first evidence of a role for ent2 function in Drosophila and suggest that the observed defects in associative learning and synaptic function may be attributable to changes in adenosine receptor activation.

Cholesterol-dependent kinase activity regulates transmitter release from cerebellar synapses.

Changes in membrane cholesterol content can alter protein kinase activity, however, it is not known whether kinases regulating transmitter release are sensitive to membrane cholesterol content. Here we have used the cholesterol extracting agent methyl-beta-cyclodextrin to measure the effects of acute cholesterol reduction on transmitter release from cultured cerebellar neurons. Cholesterol depletion increased the frequency of spontaneous transmitter release without altering the amplitude and time course of mEPSCs. Evoked transmitter release was decreased by cholesterol extraction and the paired pulse ratio was also decreased. Alterations in synaptic transmission were not associated with failure of action potential generation or changes in presynaptic Ca(2+) signaling. Both the increase in mEPSC frequency and the change in paired pulse ratio were blocked by the broad spectrum protein kinase inhibitor staurosporine. The increase in mEPSC frequency was also sensitive to selective inhibitors of PKC and PKA. Our results therefore demonstrate that the activity of presynaptic protein kinases that regulate spontaneous and evoked neurotransmitter release is sensitive to changes of membrane cholesterol content.

Peptide-mediated transdermal delivery of botulinum neurotoxin type A reduces neurogenic inflammation in the skin.

Release of inflammatory pain mediators from peripheral sensory afferent endings contributes to the development of a positive feedback cycle resulting in chronic inflammation and pain. Botulinum neurotoxin type A (BoNT-A) blocks exocytosis of neurotransmitters and may therefore block the release of pain modulators in the periphery. Subcutaneous administration of BoNT-A (2.5, 5 and 10U) reduced plasma extravasation (PE) caused by electrical stimulation of the saphenous nerve or capsaicin in the rat hindpaw skin (ANOVA, Post hoc Tukey, p<0.05, n=6). Subcutaneous BoNT-A also reduced blood flow changes evoked by saphenous nerve stimulation (ANOVA, Post hoc Tukey, p<0.05, n=6). Subcutaneous BoNT-A had no effect on PE induced by local injection of substance P (SP) or vasodilation induced by local CGRP injection. Although BoNT-A is an effective treatment for a wide range of painful conditions, the toxin's large size necessitates that it be injected at numerous sites. We found that a short synthetic peptide (TD-1) can facilitate effective transdermal delivery of BoNT-A through intact skin. Coadministration of TD-1 and BoNT-A to the hindpaw skin resulted in a significant reduction in PE evoked by electrical stimulation. The findings show that BoNT-A can be administered subcutaneously or topically with a novel transdermal delivery peptide to reduce inflammation produced by activating nociceptors in the skin. Peptide-mediated delivery of BoNT-A is an easy and non-invasive way of administering the toxin that may prove to be useful in clinical practice.

Sex differences in inflammation evoked by noxious chemical, heat and electrical stimulation.

Neurogenic inflammation (NI) is a feature of several inflammatory pain conditions in which females are overrepresented. Therefore, we asked if there are sex differences in the inflammatory response evoked by well known neurogenic stimuli. We compared the amount of plasma extravasation (PE), a measure of inflammation, in the hindpaw skin of male and female rats caused by subcutaneous injection of capsaicin, application of noxious heat (51 degrees C water bath) or electrical stimulation of the saphenous nerve. We also compared the amount of PE in males and females evoked by substance P (SP), the principal neurogenic mediator of PE. PE was quantified using a video camera and digital image analysis to measure changes in reflectance (pixel intensity, PI) of skin due to accumulation of extravasated Evans blue (EB) dye. The increase in PI induced by capsaicin was significantly greater in females compared to males (p<0.001) and in estrus, diestrus, and metestrus females compared to proestrus females. The time to reach maximal capsaicin-induced PE was two times longer in estrus, diestrus, and metestrus females compared to males (p<0.05). PE induced by heat was also significantly greater in females compared to males (p<0.001), however, there was no sex-related difference in PE induced by electrical stimulation or by injection of SP. These findings show that females have a greater inflammatory response when inflammation is induced by capsaicin and noxious heat suggesting possible sex-related changes in TRPV-1 receptor mediated mechanisms. These results add to the growing list of sex difference responses to noxious somatic stimulation.

Calcineurin and cytoskeleton in low-frequency depression.

Transmitter release at high probability phasic synapses of crayfish neuromuscular junctions depresses by over 50% in 60 min when stimulated at 0.2 Hz. Inhibition of the protein phosphatase calcineurin by intracellular pre-synaptic injection of autoinhibitory peptide inhibited low-frequency depression (LFD) and resulted in facilitation of transmitter release. Since this inhibitor had no major effects when injected into the post-synaptic cell, only pre-synaptic calcineurin activity is necessary for LFD. To examine changes in phosphoproteins during LFD we performed a phosphoproteomic screen on proteins extracted from motor axons and nerve terminals after LFD induction or treatment with various drugs that affect kinase and phosphatase activity. Proteins separated by PAGE were stained with phospho-specific/total protein ratio stains (Pro-Q Diamond/SYPRO Ruby) to identify protein bands for analysis by mass spectrometry. Phosphorylation of actin and tubulin decreased during LFD, but increased when calcineurin was blocked. Tubulin and phosphoactin immunoreactivity in pre-synaptic terminals were also reduced after LFD. The actin depolymerizing drugs cytochalasin and latrunculin and the microtubule stabilizer taxol inhibited LFD. Therefore, dephosphorylation of pre-synaptic actin and tubulin and consequent changes in the cytoskeleton may regulate LFD. LFD is unlike long-term depression found in mammalian synapses because the latter requires in most instances post-synaptic calcineurin activity.Thus, this simpler invertebrate synapse discloses a novel pre-synaptic depression mechanism.