Strong and independent associations between plasma concentration of cystatin C and risk of cardiovascular disease (CVD) suggests causal involvement of cystatin C.The aim of our study was to assess whether there is a causal relationship between plasma concentration of cystatin C and risk of coronary artery disease (CAD) using a Mendelian Randomization approach.We estimated the strength of association of plasma cystatin C on CAD risk and the strength of association of the strongest GWAS derived cystatin C SNP (rs13038305) on plasma cystatin C in the population-based Malmö Diet and Cancer Study (MDC) and thereafter the association between rs13038305 and CAD in the MDC (3200 cases of CAD and 24418 controls) and CARDIOGRAM (22233 cases of CAD and 64762 controls).-35), resulting in a power of >98% to detect a significant relationship between rs13038305 and CAD in MDC and CARDIOGRAM pooled. The odds ratio for CAD  was 1.00 (0.94–1.07); P = 0.92 in MDC, 0.99 (0.96–1.03); P = 0.84 in CARDIOGRAM and 1.00 (0.97–1.03); P = 0.83 in MDC and CARDIOGRAM pooled.Each standard deviation (SD) increment of plasma cystatin C was associated with increased risk of CAD  after full adjustment. Each copy of the major allele of rs13038305 was associated with 0.34 SD higher plasma concentration of cystatin C (P<1 x 10Genetic elevation of plasma cystatin C is not related to altered risk of CAD, suggesting that there is no causal relationship between plasma cystatin C and CAD. Rather, the association between cystatin C and CAD appears to be due to the association of eGFR and CAD. Human γ-trace, also called post-γ-globulin, was first described in 1961 and the name Cystatin C was proposed in 1984–4. CystaCystatin C, a non-glycosylated cationic low molecular weight protein Mw 13 343 Da) is an inhibitor of cysteine proteinases. This protein is eliminated by glomerular filtration, reabsorbed and catabolized in proximal renal tubular cells without tubular secretion, 9 and t 343 Da iIncreased plasma level of cystatin C is associated with increased risk of cardiovascular disease (CVD) and mortality in the elderly –16 and iGenome-wide Association studies (GWAS) for genetic determinants of cystatin C have pinpointed the strongest signal of the genome associating with variation of plasma concentration of cystatin C at the cystatin C locus on chromosome 20, represented by the single nucleotide polymorphism (SNP) rs13038305. Of note, this SNP was not associated with creatinine-based measures of renal function and thus likely to affect cystatin C plasma concentration independently of renal function .We hypothesized that cystatin C may exert GFR independent effects increasing the risk of coronary artery disease (CAD) and thus set out to test whether or not there is a causal relationship between cystatin C and risk of CAD using a Mendelian Randomization approach where the strongest genetic signal for plasma concentration of cystatin C identified thus far (rs13038305) was related to risk of CAD.The Malmö Diet and Cancer study (MDC) is a population-based prospective cohort consisting of 30 447 subjects (DNA available on n = 28 767) surveyed at a baseline examination in 1991–1996 . From thDNA was extracted from frozen granulocytes or buffy coats with the use of QIAamp-96 spin blood kits  at the DNA extraction facility supported by SWEGENE. We successfully genotyped the plasma cystatin C associated SNP rs13038305 at the cystatin C locus on chromosome 20 in 27 618 subjects of the MDC. Primers and probes were custom synthesized by Applied Biosystems  according to standard recommendations for the AB Prism 7900HT analysis system, and genotyped with polymerase chain reaction-based TaqMan method.2) = (140-age) x weight in kg x 1.23 / P-creatinine  (CG)[2 by estimating BSA for all persons from the duBois formulae (BSA in m2 = 0.00718 x (length in cm)0,725 x (weight in kg)0,425). MDRD was calculated from the four-variable MDRD GFR formulae (mL/min/1.73 m2) = 175 x (P-creatinine / 88.4) −1.154 x age −0.203 [Within the subsample of MDC-CC we analysed plasma concentration of cystatin C and creatinine in fasting plasma samples in 4743 subjects who also completed genotyping of the rs13038305. Levels of plasma cystatin C were measured using a particle-enhanced immunonephelometric assay  and presented in mg/L. Plasma f female). AdjustmAll MDC participants underwent a medical history and physical examination. Blood pressure was measured using a mercury-column sphygmomanometer after 10 minutes of rest in the supine position. Hypertension was defined as systolic or diastolic blood pressure ≥140/90 mmHg or use of antihypertensive medication. Diabetes mellitus was defined as a fasting whole blood glucose >109 mg/dl (6.0 mmol/L), a self-reported physician diagnosis of diabetes, use of anti-diabetic medication or having been diagnosed in local or national Swedish diabetes registries, as described previously . CigaretWithin the MDC-CC we measured fasting total cholesterol, HDL cholesterol, and triglycerides according to standard procedures at the Department of Clinical Chemistry, University Hospital Malmö. LDL cholesterol was calculated according to Friedewald’s formula.Retrieval of prevalent (occurring before the baseline exam of the MDC) and incident (occurring after the baseline exam of the MDC) cases of CAD were identified through linkage of the 10-digit personal identification number of each Swedish citizen with three registers: the Swedish Hospital Discharge Register, Swedish Coronary Angiography and Angioplasty Registry (SCAAR) and the Swedish Cause of Death Register. The registers have been previously described and validated for classification of outcomes , 34. FolCAD was defined as fatal or non-fatal myocardial infarction (MI), death from ischemic heart disease, CABG or PCI, whichever came first. MI was defined on the basis of International Classification of Diseases 9th and 10th Revisions (ICD9 and ICD10) codes 410 and I21, respectively. Death due to ischemic heart disease was defined on the basis of codes 412 and 414 (ICD9) or I22–I23 and I25 (ICD10). CABG was identified from national Swedish classification systems of surgical procedures, the KKÅ system from 1963 until 1989 and the Op6 system since then. CABG was defined as a procedure code of 3065, 3066, 3068, 3080, 3092, 3105, 3127, 3158 (Op6) or FN (KKÅ97). PCI was defined based on the operation codes FNG05 and FNG02.All MDC participants gave informed consent and the study was approved by the local ethics committee, Lund University, Sweden (LU-51-90).The CARDIoGRAM study is a meta-analysis of 14 GWAS studies of CAD comprising 22 233 individuals with CAD and 64 762 controls of European descent . IncludeOf note, 185 participants (86 CAD cases and 99 control subjects) of the MDC were also participants in the Myocardial Infarction Genetics Consortium , later iWe first estimated the strength of association of the association between plasma cystatin C (expressed as per 1 SD increase) and risk of incident CAD, after exclusion of prevalent CAD cases, using logistic regression in the MDC-CC. Cystatin C was transformed with the natural logarithm before analysis due to skewness to the right of its distribution. Two models adjusting for age and sex (model 1) and full risk factor adjustment , were applied. Thereafter, again in the MDC-CC, we used linear regression to test the association between the rs13038305  and standardized values of LN-transformed cystatin C (dependent variable) adjusting both according to model 1 and model 2 (see above). Based on the plasma cystatin C vs. CAD and the rs13038305 vs. plasma cystatin C relationships we then calculated the power to detect association between rs13038305 and CAD in MDC + CARDIoGRAM, assuming that the rs13038305 genetic effect on plasma cystatin C would be independent of renal function and that the relationship of the rs13038305 genetic plasma cystatin C elevation on CAD would be proportional to the over-all relationship between plasma cystatin C and CAD.Finally, we performed logistic regressions testing the association between rs13038305 and CAD  in a model adjusted for age and sex, in the entire MDC (3200 CAD cases and 24418 control subjects) and in CARDIoGRAM (22 233 CAD cases and 64 762 controls subjects) and meta-analysed the results of the two studies.SPSS statistical software  was used for all calculations except for the power calculations which were performed using the PS Power and Sample Size Calculations software version 3.0  and the meta-analysis was performed by Inverse-variance-weighted fixed effects model using STATA 11 .The clinical characteristics of the MDC and in the subsample of MDC-CC (in whom plasma cystatin C was available) are shown in -6) after age and sex adjustment and 1.20, 1.07–1.34 (P = 0.001) after full risk factor adjustment according to model 2.Among MDC-CC subjects free from CAD at the baseline exam , 414 subjects developed CAD during follow-up. Each standard deviation (SD) increment of plasma cystatin C was significantly related to increased risk of CAD during follow-up  1.28, 1.16–1.42 (P = 1.8x10−35) approximately one third of a SD increase of plasma concentration of cystatin C both in model 1 and model 2. On the contrary, there was no significant association between rs13038305 and creatinine based estimation of renal function (CG and MDRD)  .At an α = 0.05 and based on the plasma cystatin C vs CAD and rs13038305 vs plasma cystatin C relationships we had 100% power to detect an association between rs13038305 and CAD in the pooled MDC + CARDIoGRAM analysis if we applied the model 1 (age and sex adjusted) effect estimates from the plasma cystatin C vs CAD (OR = 1.28 for CAD per 1 SD increase of cystatin C) whereas the corresponding power estimate was 98% if we applied the model 2  effect estimates from the plasma cystatin C vs CAD (OR = 1.20 for CAD per 1 SD increase of cystatin C). However, among the total of 25 433 CAD cases and 89 081 control subjects, the rs13038305 had an odds ratio of 1.00  in the pooled analysis and similar strength of association in the two studies analysed separately .We here tested whether there is a causal relationship between cystatin C and risk of CAD using a Mendelian randomization approach by testing whether genetic elevation of cystatin C, measured by a SNP at the cystatin C locus on chromosome 20 robustly associated with plasma cystatin C (rs13038305), alters the risk of CAD in more than 25 000 cases of CAD and almost 90 000 control subjects. Our main finding is that genetic elevation of cystatin C, which in contrast to over-all plasma concentration of cystatin C is not confounded by environmental exposures, is not associated with altered risk of CAD. This strongly implies that cystatin C does not have a causal relationship with risk of CAD. Thus the epidemiological relationship between plasma cystatin C and CAD, despite being statistically independent from CVD risk factors, is likely to be explained by correlation between cystatin C and long-term exposure to CVD risk factors such as hypertension, diabetes and impaired renal function.per se in CAD development, selection of the rs13038305 SNP at the cystatin C locus was particularly important mainly for two of reasons. First, as compared with genetic effects on many other previously examined cardiovascular biomarkers, the rs13038305 has a comparatively large effect on its circulating gene product, resulting in approximately one third of a SD increase of plasma cystatin C per copy of the major allele, thus favouring the power of our study. Second, previous studies [When examining whether or not there may be a causal role for cystatin C  studies  and our It should be emphasized that the function of rs13038305 and the causal relation between rs13038305 and cystatin C are unknown. It is possible that rs13038305 or the causal variant(s) that it represents may influence more than one trait with opposite effects on CAD. This would also result in lack of association between rs13038305 and CAD.When calculating the power of our Mendelian Randomization study, we based the expected genetic cystatin C elevating effect on CAD on the epidemiological relationship between plasma cystatin C and CAD development. There are several considerations and assumptions linked to such a power calculation. First, if cystatin C itself would mediate detrimental effects on CAD which acted through CVD risk factors, the effect estimate derived from model 1 (age and sex adjusted only) would be most appropriate which would mean that we had close to 100% power to find an association between rs13038305 and CAD. In contrast, if the genetic effect were completely unrelated to CVD risk factors, it would have been more appropriate to apply the effect estimate obtained from model 2 (fully adjusted for CVD risk factors) which resulted in a power of 98% to link genetic effects of cystatin C to CAD risk. We took into account both of these two scenarios and we believe that even the lower power estimate of 98% is sufficient to conclude with relatively large certainty, although not with one hundred percent, that our results exclude a causal relationship between cystatin C and CAD risk.Although all epidemiological studies point at direct relationship between elevated cystatin C and risk of CVD, there is mechanistic evidence that cystatin C could in fact be protective by inhibiting cathepsins and thus theoretically to reduce matrix degradation and vascular structure and function . This haGenetic elevation of plasma cystatin C is not related to altered risk of CAD, suggesting that there is no causal relationship between plasma cystatin C and altered risk of CAD. Our results suggest that high level of cystatin C before CAD occurs as result of co-variation with CVD risk factors and impaired renal function. The lack of a causal relationship does not encourage development of interventions targeted at the cystatin C system in attempts to prevent CAD.


Transgenic mice ubiquitously expressing a surrogate UPS substrate (GFPdgn) were treated with bafilomycin-A1 (BFA) to inhibit the ALP. Myocardial and renal GFPdgn protein levels but not mRNA levels were increased at 24 hours but not 3 hours after the first injection of BFA. Myocardial protein abundance of key proteasome subunits and the activities of proteasomal peptidases were not discernibly altered by the treatment. In cultured neonatal rat ventricular myocytes (NRVMs), the surrogate UPS substrate GFPu and a control red fluorescence protein (RFP) were co-expressed to probe UPS performance. At 12 hours or 24 hours after ALP inhibition by 3-methyladenine (3-MA) or BFA, GFPu/RFP protein ratios and the protein half-life of GFPu were significantly increased, which is accompanied by increases in p62 proteins. Similar findings were obtained when ALP was inhibited genetically via silencing Atg7 or Rab7. ALP inhibition-induced increases in GFPu and p62 are co-localized in NRVMs. siRNA-mediated p62 knockdown prevented ALP inhibition from inducing GFPu accumulation in NRVMs. We conclude that in a p62-dependent fashion, ALP inhibition impairs cardiac UPS proteolytic performance in cardiomyocytes in vitro and in vivo.Intracellular protein degradation is primarily performed by the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP). The interplay between these two pathways has been rarely examined in intact animals and the mechanism underlying the interplay remains unclear. Hence, we sought to test  The degradation of most proteins, native or misfolded, in the cell is carried out by the ubiquitin-proteasome system (UPS). UPS-mediated protein degradation involves two main steps: ubiquitination of individual substrate protein molecules and degradation of the ubiquitinated proteins by the proteasome The autophagic-lysosomal pathway (ALP) represents another major cytoplasmic catabolic pathway that delivers cytoplasmic components (including organelles) to the lysosome for degradation in vivo effect of inhibition of the ALP on UPS function. The few reported studies, which used only cell cultures, came to contradictory conclusions. Korolchuk et al. showed in cultured HeLa cells and mouse embryonic fibroblasts (MEF) that pharmacological or genetic inhibition of autophagy does not alter proteasome peptidase activities but hinders the degradation of ubiquitinated substrates by the proteasome due to accumulation of p62/SQUSTM1 et al. reported that inhibition of autophagy leads to increased proteasome activities and upregulation of proteasomal subunits in cultured human colon cancer cells under nutrient-deficient conditions The proteasomal and lysosomal degradation were historically regarded as two parallel pathways but emerging evidence suggests that the two pathways actually interact with each other and their interplay may be critical to maintaining proteostasis in the cell In the present study, we have examined the impact of inhibition of the ALP at the autophagosome formation or removal stages on UPS proteolytic function using a mouse model of an inverse reporter of UPS function. We also investigated the effect of the inhibition of the ALP on UPS function in cultured cardiomyocytes. Our results indicate that inhibition of the ALP causes a delayed impairment of UPS performance in a p62-dependent manner in cardiomyocytes.This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . It was also approved by the Institutional Animal Care and Use Committee of the University of South Dakota, Vermillion, SD, USA. The creation and initial characterization of transgenic (tg) mice which constitutively and ubiquitously express an enhanced green fluorescence protein (GFP) that is modified by carboxyl fusion of degron CL1 (the fusion GFP is known as GFPdgn) have been previously described The GFPdgn tg mice of approximately 3 months of age were subject to intraperitoneal injections of bortezomib , bafilomycin A1 , or vehicle control . The mice tolerated the treatment well and no gross abnormality was observed. At 3 hours after BZM injection and at 3 and 24 hours after the first BFA or DMSO injection, the mice were sacrificed through deep anesthesia via isoflurane inhalation and subsequent heart removal. Ventricular and renal tissues were then sampled for total protein and RNA extractions or fixed with 3.8% paraformaldehyde.Neonatal rats at postnatal day 0 to day 2 were sacrificed by decapitation and subsequent heart removal. Ventricular myocytes were then isolated using the Cellutron Neomyocytes isolation system  following the manufacturer's instructions Creation and validation of recombinant adenoviruses (Ad-) harboring the expression cassette for a red fluorescence protein (RFP) or a GFP with carboxyl fusion of the degron CL1 (GFPu) were previously reported This was performed as previously described These assays were done as previously described 5′-TCT ATA TCA TGG CCG ACA AGC AGA-3′ and reverse 5′-ACT GGG TGC TCA GGT AGT GGT TGT-3′; GAPDH: forward 5′-GCC GTA TTG GGC GCC TGG TCA-3′ and reverse 5′-AAC ATA CTC AGC ACC GGC CTT ACCC-3′. Relative mRNA levels were normalized with GAPDH mRNA levels.Total RNA was extracted from ventricular myocardium using the Tri-Reagent  as previously described These were performed as previously described NRVMs grown on the cover glass were fixed with 3.8% paraformaldehyde for 10 minutes, permeabilized with 1% of Triton-X100 in PBS for 1 hour, quenched with 0.1 M glycine in PBS for 1 hour, and blocked with 0.5% BSA for 1 hour. The specimens were then incubated with primary antibodies overnight at 4°C. The antibody against p62  was used along with Alexa-Fluor 568 donkey anti-guinea pig Ig (Molecular Probes) to immunofluorescence label p62 in the cell. The GFPu direct fluorescence and p62 indirect fluorescence were captured using the fluorescence confocal microscope.This was performed as previously described 5′- CATGTCCTATGTGAAAGATGA-3′), Atg7 (siAtg7: Cat. # SI01729119), Rab7 (siRab7: Cat. # SI03021725) and the siRNA targeting luciferase serving as a control siRNA (siLuc: 5′-AACGTACGCGGAATACTTCGA-3′) were all purchased from Qiagen . LipofectamineTM 2000 transfection reagent (Invitrogen) was used for siRNA transfection following the manufacturer's protocol. Transfection of cultured NRVMs with siRNA was generally started at 48–72 hours after NRVMs were plated. In p62 knock down experiments, the same amounts of luciferase siRNA or p62 siRNA were applied to the control and experimental groups, respectively. Six hours after the transfection, the siRNA-containing medium was replaced with the fresh medium containing 2% FBS. To effectively knock down p62 protein levels, 2 consecutive rounds of transfection of p62 siRNA at a dose of 160 pmol for 2×106 cells with an interval of 72 hours were performed The siRNA's specific for rat p62 ; by contrast, myocardial GFPdgn protein levels remained unchanged at 3 hours after the administration of BFA. The effective inhibition of lysosomes by BFA is illustrated by the increases in LC3-II and p62 in the heart of BFA-treated mice. Importantly, myocardial and renal GFPdgn protein levels were significantly increased at 24 hours after the first injection of BFA . The GFPdgn protein increases were not accompanied by discernible alteration in the level of steady state GFPdgn mRNA in either cardiac or renal tissues  or the 20S proteasomes  in ventricular myocardium were measured using western blot analyses. The protein abundance of the proteasome subunits was found comparable among the CTL, BZM-3h, BFA-3h, and BFA-24h groups . Moreover, both 3-MA and BFA significantly increased the GFPu/RFP ratio at 24 h of treatment (see next section).To test whether the effect of ALP inhibition on the degradation of a UPS substrate in the heart is cardiomyocyte-autonomous and is not a secondary response to altered heart function at the organ level or to a systemic neurohumoral factor, we employed a NRVM culture system to investigate the effect of pharmacological or genetic inhibition of the ALP on the UPS. Pharmacologically, 3-methyladenine (3-MA) was used to inhibit the initiation step of macroautophagy or autophagosome formation and bafilomycin A1 (BFA), which inhibits autophagosome-lysosome fusion and lysosomal function, was utilized to inhibit autophagosome removal. UPS performance was evaluated using the GFPu/RFP ratio. Similar to GFPdgn, GFPu is a proven efficient substrate of the UPS;To further verify that GFPu is stabilized by a long-term treatment of 3-MA or BFA, we performed the cycloheximide (CHX) chase assay in the cultured NRVMs. This assay allows monitoring the degradation rate of GFPu when protein synthesis is blocked by CHX. CHX administration was initiated at 12 h after ALP inhibition was started. The protein levels of GFPu and RFP in the total cell lysates immediately before (0 min) or at 5, 10, 15, 30, 60, and 120 min after CHX administration were measured via western blot analyses. These assays showed that after 12 h of treatment of either 3-MA or BFA, the degradation of GFPu was significantly slowed down whereas the degradation of RFP was not discernibly affected Figure 5in vivo and in vitro.Taken together, these experiments demonstrate that long-term (12 h or longer) but not short term (<6 h) inhibition of the ALP impairs the degradation of proteasome substrates in cardiomyocytes Given that the accumulation of p62, a protein capable of binding ubiquitinated proteins, occurs before GFPu stabilization becomes discernible during ALP inhibition Figure 4The product of the autophagy-related gene 7 (Atg7) plays an important role in the formation of autophagosomes and the ablation of Atg7 impairs macroautophagy The effect of ALP inhibition on UPS performance in intact animals has rarely been investigated. Using cultured mammalian non-cardiomyocyte cells, two studies reported so far have yielded opposing results concerning the impact of ALP inhibition on proteasomal function et al. reported similar findings in cultured Hella cells and mouse embryonic fibroblasts using a different surrogate proteasome substrate in vivo impact of lysosomal malfunction on proteasomal function was reported by Qiao et alUPS-mediated protein degradation is an ATP-dependent sophisticate process that requires the collaboration of a large number of proteins involved in ubiquitination of the substrate protein, delivery of the ubiquitinated proteins to the proteasome, and proteasomal degradation bona fide UPS substrate  and an LC3 interacting motif (LIR), p62 is purported to target aberrant protein aggregates and defective organelles  which carry poly-ubiquitin moieties suitable for UBA to bind, to autophagosomes for ALP-mediated removal Presently, it is unclear how exactly p62 accumulation impairs UPS performance in cardiomyocytes but p62 is known to bind polyubiquitinated proteins via its UBA domain and to self-oligomerize via its PB1 domain Taken together, our data suggest that long-term inhibition of the ALP impairs the degradation of UPS substrates in a p62 dependent manner. The implication of this discovery is potentially broad and significant for both basic and translational research. For example, this may help explain why most lysosomal storage diseases are precipitating in nature; evidence of UPS malfunction and lysosomal deficiency was found to co-exist in failing human hearts


Targeting BCR/ABL with Tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias but the “gatekeeper” mutation T315I confers resistance against all approved TKIs, with the only exception of ponatinib, a multi-targeted kinase inhibitor. Besides resistance to TKIs, T315I also confers additional features to the leukemogenic potential of BCR/ABL, involving endogenous BCR. Therefore we studied the role of BCR on BCR/ABL mutants lacking functional domains indispensable for the oncogenic activity of BCR/ABL. We used the factor independent growth of murine myeloid progenitor 32D cells and the transformation of Rat-1 fibroblasts both mediated by BCR/ABL. Here we report that T315I restores the capacity to mediate factor-independent growth and transformation potential of loss-of-function mutants of BCR/ABL. Targeting endogenous Bcr abrogated the capacity of oligomerization deficient mutant of BCR/ABL-T315I to mediate factor independent growth of 32D cells and strongly reduced their transformation potential in Rat-1 cells, as well as led to the up-regulation of mitogen activated protein kinase (MAPK) pathway.Our data show that the T315I restores the capacity of loss-of-function mutants to transform cells which is dependent on the transphosphorylation of endogenous Bcr, which becomes a putative therapeutic target to overcome resistance by T315I. The fine regulation of ABL kinase is lost by its fusion to BCR in the context of the t9;22). The constitutive activation of the ABL-kinase activity induces aberrant proliferation and neoplastic transformation by constitutive activation of down-stream signaling pathways such as RAS, PI3 kinase, or STATs , 2. Seve2. The coThe inhibition of aberrant BCR/ABL kinase activity by selective ATP mimetics (TKI) such as imatinib, dasatinib or nilotinib, is a valid concept of causal therapy for Ph+ leukemia -11. UnfoThe “gatekeeper” mutation T315I confers not only resistance against these ATP-competitors but also against some allosteric inhibitor, such as GNF-2 or against inhibition of oligomerization . The onlThe mechanisms by which T315I confers resistance to TKI were mainly attributed to an alteration of the three-dimensional structure of the ATP-binding site, leading to a loss of a fundamental H bond -25. MoleThe key event for the activation of the BCR/ABL kinase is the tetramerization mediated by the N-terminal coiled coil (CC) region of the BCR-portion , 30. AccNoteworthy factor independent growth was suppressed by the inhibition of tetramerization in hematopoietic progenitors expressing BCR/ABL harboring the Y253F and E255K mutations but not the T315I mutation , 28. In In order to further confirm the role of the endogenous BCR for the mediation of resistance mediated by the T315I we investigated further the effect of T315I on the transformation potential and the capacity to mediate factor independent growth of BCR/ABL mutants lacking functional domains in the BCR portion considered indispensable for the oncogenic activity of BCR/ABL.BCR/ABL but not of p185-T315IBCR/ABL. This strongly suggests that the “gatekeeper“ mutation T315I is not only responsible for TKI-resistance, but also confers additional properties to BCR/ABL which is uncovered in the case of a loss of oligomerization.Targeting oligomerization either by competitive peptides or by deleting the CC-domain efficiently interferes with the transformation potential of native p185BCR/ABL was used as a control.To further disclose the influence of T315I on the biology of BCR/ABL we investigated the effects of T315I on “loss of function mutants” of BCR/ABL, unable to mediate factor independent growth of murine hematopoietic progenitors. The schematic representation of the mutants used in this study is shown in Figure BCR/ABL, which was completely restored by the presence of T315I.The phosphorylation at Y177 is indispensable for the function of BCR/ABL. To investigate the effects of T315I on a mutant defective in phosphorylation at Y177 we used p185-T315I-Y177F in which Y177 was mutated to phenylalanine. As shown in Figure Next we focused on the influence of T315I on BCR/ABL with deficiencies in oligomerization. As reported in Figure In order to confirm that both oligomerization interface and Y177 phosphorylation site are dispensable for the function of T315I, we used a combined mutant - ΔCCp185-Y177F - lacking both the CC oligomerization interface and the phosphorylation site at Y177. Even in this mutant, which lacks two functions considered to be indispensable for the activity of BCR/ABL and whose deletion abolished factor independent growth, T315I restored the capacity to confer factor independent growth Figure .BCR/ABL construct in which the entire S/T domain was deleted with or without the T315I. The deletion of S/T domain attenuated the factor independent growth of 32D cells mediated by p185BCR/ABL, which was restored by the presence of T315I to the level of native p185BCR/ABL  domain of BCR we investigated the effects of T315I on the factor independent growth of 32D cells expressing a p185L Figure .BCR/ABL(#ABL)  Figure .BCR/ABL, which allow to restore the capacity to mediate factor independence in 32D cells.Taken together, these data indicate that the “gatekeeper” mutation T315I confers additional properties to the loss of function mutants of p185BCR/ABL, we performed classical transformation assays in order to study both the loss of contact inhibition and anchorage-dependent growth in retrovirally transduced Rat-1 fibroblasts, by focus and colony formation assays, respectively.Factor independent growth of 32D cells stands for the substitution of the IL-3 signaling by an alternative survival signal, which can be mediated by activated kinases such as BCR/ABL. To investigate the influence of T315I on the transformation potential of loss-of function mutants of p185BCR/ABL expressing Rat-1 cells  is shown in Figure BCR/ABL, whereas it abrogated the capacity of ΔCC-p185-T315I to mediate factor independent growth  to disclose role of the endogenous BCR for the new biological features determined by T315I in p185ll model , 31, 32.Ph+ leukemia, neither CML nor Ph+ ALL, ever emerges with a full blown T315I-positive leukemia even if the clone with the mutation is already existing and detectable . TherefoHow T315I is modulating the function of BCR/ABL is nearly unknown, mainly due to the fact that actually available models do not clearly reveal differences. Therefore we employed loss of function mutants of native BCR/ABL in which T315I restores at least parts of the BCR/ABL related phenotype.T315I seems to bypass the autophosphorylation at Y177 as the top of the RAS/ERK-1/2-signalling cascade initiated by BCR/ABL. Its presence leads to an activation of the RAS-signalling even in the absence of a fully functional BCR/ABL kinase. In native BCR/ABL phosphorylation at Y177 provides a docking site for the adapter molecule GRB-2, which stabilizes RAS in its active GTP-bound form by the binding to SOS , 35. ActOur data suggest that the transphosphorylation of BCR seen in the presence of the T315I substitutes for the role of the Y177 in BCR/ABL itself and adopted by the endogenous BCR. This is confirmed by our findings that targeting endogenous Bcr by RNAi not only abolished the effects of T315I on Erk1/2 activation, but also the transformation potential and factor independent growth of ΔCC-p185-T315I in murine 32D cells. Translated this scenario into a patient harboring a BCR/ABL-T315I would mean that in the absence of treatment an additional activation of the RAS/MAPK-pathway may also interfere with the proliferation or even senescence , 37, wheBCR/ABL, p210BCR/ABL transactivates BCR. In fact it co-precipitates BCR from Ph+ cell lines and is a target for the BCR/ABL tyrosine kinase activity [The mechanism by which the presence of T315I leads to trans-phosphorylation of BCR by BCR/ABL loss of function mutants remains to be clarified. Similar to our findings on p185activity , 38-41. activity , 42.BCR/ABL was able to restore only factor independence but not full transformation potential indicating that it needs different domains for different phenotypes like factor independence and transformation potential which might be responsible for the additional oncogenic activity of T315.Another important aspect of our study is the fact that we can now distinguish between features important for the mediation of factor independent growth and those important for the classical transformation potential. The combined deletion of the serine/threonine (S/T) and Grb-2 binding (Y177) domains completely abolished the transformation potential of p185 - T315IIn summary, our data show that T315I not only interferes with the affinity of ATP-analogs for the ABL-kinase, but also conferring strong biological activity that seems to be related to the phosphorylation of endogenous bcr. These findings introduce new approaches for the molecular therapy of patients harboring TKI-resistant BCR/ABL mutations.BCR/ABL, p185-T315, their respective mutants lacking the N-terminal CC domain (ΔCC-p185 and ΔCC-p185-T315I), the ABL part of BCR/ABL (#ABL and #ABL-T315I), deletion mutants of putative BCR serine/threonine kinase domain BCR(1-196)/ABL and BCR(1-196)/ABL-T315), the constructs with a direct fusion of the CC-domain of BCR to the ABL-portion of BCR/ABL (BCC-ABL and BCC-ABL-T315I), the p185-Y177F and p185-Y177F-T315I as well as the ΔCC-p185-Y177F and ΔCC-p185-Y177F-T315I and the related retroviral PINCO vectors have been previously described [The cDNAs encoding p185escribed , 30. Sho32D cells were obtained from the German Collection of Microorganisms and Cell Cultures  and maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) (Invitrogen) containing 10 ng/ml IL-3 . Ecotropic Phoenix cells, 293T cells and Rat-1 cells were cultured in DMEM supplemented with 10% FCS.BCR/ABL. Six-well plates were filled with DMEM supplemented with 10% FCS and 0.5% bacto-agar . Then, 5×103 transduced Rat-1 cells were suspended in ‘top-agar’ (DMEM supplemented with 10% FCS and 0.25% bacto-agar) and stacked in the wells. Colonies were counted after 15 days incubation. For focus-formation assays in a 24-well plate format, 4×104 transduced Rat-1 cells were plated per well. Foci were stained after 15 days using 1% crystal violet (Sigma-Aldrich).Soft-agar and focus formation assays were performed using Rat-1 fibroblasts retrovirally transduced with PINCO vectors harboring native or mutant p1855 target cells were plated onto retronectin-coated  24-well-plates and exposed to the retroviral or lentiviral supernatant and were centrifuged at 2.200 rpm for 45 min on 32°C. The infection was repeated 3-4 times and infection efficiency was measured after 48 h by determining the percentage of GFP positive cells by flow cytometry.Ecotropic retroviral supernatants were obtained as described earlier . LentiviWestern blot analyses were performed according to widely established protocols. The following antibodies were used: anti-ABL (a-ABL) Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylated ABL specific for the phosphorylated tyrosine-residues 245 (a-p-ABL-Y245) , anti-BCR (a-BCR) (Santa Cruz Biotechnology), anti-phosphorylated BCR-Y177 (a-p-BCR-Y177) , anti-STAT5, anti phosphorylated STAT5 , anti-Erk1/2 and anti phosphorylated Erk1/2 . Blocking and antibody incubation were performed in 5% low-fat dry milk.Proliferation was assessed by using the XTT proliferation kit , according to the manufacturer's instructions. Apoptosis was measured by 7-AAD staining as described previously .


In 3‐day organ culture, PF‐4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high‐K+ (60 mmol/L) or to α1‐adrenergic stimulation by cirazoline. Basal and stretch‐induced PYK2 phosphorylation in culture were inhibited by PF‐4594755, closely mimicking inhibition of non‐voltage‐dependent calcium influx by 2‐APB (30 μmol/L). In contrast, the L‐type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch‐induced but not basal PYK2 phosphorylation. Stretch‐induced Akt and ERK1/2 phosphorylation was eliminated by PF‐4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch‐sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 μmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage‐ and non‐voltage‐dependent calcium influx. A small‐molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle.Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium‐dependent signals. We studied the role of the calcium‐ and integrin‐activated proline‐rich tyrosine kinase 2 (PYK2) in stretch‐induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr‐402 was increased both by a 10‐min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF‐4594755  in stretch‐induced responses of the rat portal ex vivo. PYK2 phosphorylation at Tyr‐402 was increased both by a 10‐min stretch and by organ culture under stretch for 3 days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF‐4594755 (0.5–1 μmol/L), but contractile differentiation was not affected. Basal and stretch‐induced PYK2 phosphorylation in culture were inhibited by PF‐4594755, closely mimicking inhibition of non‐voltage‐dependent calcium influx by 2‐APB (30 μmol/L). In contrast, the L‐type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch‐induced but not basal PYK2 phosphorylation. The results suggest that PYK2 signaling functionally distinguishes effects of voltage‐ and non‐voltage‐dependent calcium influx. Vascular smooth muscle cells play dual roles in the vessel wall by, on one hand, regulating blood flow and, on the other, preserving the integrity of the vessel wall via proliferation, migration, and protein synthesis in response to environmental challenges such as mechanical stimuli, tissue injury, and inflammation. This dual role of smooth muscle is reflected in phenotypic plasticity enabling vascular smooth muscle to take on either a “contractile” or a “synthetic” phenotype appropriate for these different roles Owens . SustainRecent studies have largely clarified the mechanisms regulating the expression of contractile and cytoskeletal proteins, which is generally driven by serum response factor (SRF) in concert with the coactivators myocardin and myocardin‐related transcription factors . A key element is translocation of MRTF from the cytosol to the nucleus in response to decreased concentration of globular (G‐) actin  is known to be phosphorylated in response to multiple stimulants including angiotensin II (Ang II), increased intracellular calcium, and integrin activation, and to influence multiple signal systems including the MAP kinase and Rho pathways as well as ion channel activity  and the other strip unloaded. The strips were cultured in DMEM‐Hank's F12 (1:1) with 2% dialyzed FCS and 10−9 mol/L insulin as described . This investigation conforms to Directive 2010/63/EU of the European Parliament. Rats  were bought from Taconic (Denmark). They were sacrificed using COμmol/L (max. 0.08% DMSO), a concentration range showing high kinome selectivity  has been described, was dissolved in DMSO, and used at 0.5 or 1 2, 1.2 MgCl2, 11.6 HEPES; pH 7.4) and maintained at 37°C. Inhibitor or vehicle was added to the medium as appropriate. Vessels were equilibrated for 45 min after applying a basal tension of 3 mN. Following recording of spontaneous activity, strips were contracted twice with 60 mmol/L KCl‐containing HEPES medium. Following each contraction, they were relaxed in normal HEPES medium. They were then contracted with increasing concentrations of cirazoline. Contractile responses were measured by calculating the mean force over a 5‐min interval following addition of agonist . Even though the pattern of frequency and amplitude of contractions may vary between preparations and after agonist addition, mean force provides a reliable and reproducible quantitation of spontaneous activity in the portal vein. To induce maximal contractions by irreversible myosin light chain phosphorylation, calyculin A (1 μmol/L) was added in calcium‐free HEPES medium at the end of the experimental protocol.Following organ culture under load in the presence or absence of PF‐4594755, portal vein strips were mounted in a myograph  in HEPES‐buffered medium  according to the manufacturer's instructions. The relative expression of mRNA was analyzed by quantitative PCR  using QuantiFast SYBR Green RT‐PCR Kit . The following QuantiTect primer assays (Qiagen) were used: GAPDH , Tagln , Des , Acta2 , Tpm1 . Primers sequences are proprietary of Qiagen.μg) of protein were loaded in each lane of Bio‐Rad TGX 4–15% Criterion gels. Proteins were then transferred to a nitrocellulose membrane  either using a semidry transfer  or wet transfer overnight (Bio‐Rad). Proteins were detected using commercially available primary antibodies: Akt , phospho‐Akt , ERK1/2 , phospho‐ERK1/2 , FAK , purchased from Cell Signaling Technology , phospho‐FAK  and phospho‐PYK2  from Invitrogen , and PYK2  from Sigma . Secondary mouse or rabbit HRP‐conjugated antibodies  were used. Bands were visualized using ECL (Pierce West Femto) and images were acquired using the Odyssey Fc Imager .Proteins were extracted as described previously  in low‐methionine medium for the last 24 h of 3–5 day organ culture as described previously . Following thorough wash with cold PBS, they were then incubated with 10% TCA for 1 h at 4°C to allow unincorporated [3H]‐thymidine to leak out. They were subsequently washed twice with TCA and frozen in liquid nitrogen. Radioactivity was measured by liquid scintillation counting. Radioactivity was normalized to the total protein concentration in each sample.After 3 days of organ culture portal vein strips were exposed for 1 h to radio‐labeled methyl‐[P‐values were calculated by Student's t‐test for single comparisons and with two‐way analysis of variance (ANOVA) followed by Bonferroni post hoc testing for multiple comparisons involving effects of both stretch and added inhibitors. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc.). P <0.05 was considered statistically significant.Values are presented as mean ± SEM unless otherwise stated. μmol/L  3‐kinase  load‐induced increase in SM22α contents is seen . Responses to 60 mmol/L KCl‐containing HEPES buffer (high‐K+) and to α1‐adrenergic receptor stimulation by cirazoline, measured as mean force over 5 min after the addition of agonist (see Methods), were similar in control strips and in strips cultured with PF‐4594755 .To test the effect of PYK2 inhibition on contractile responses, portal venous strips were cultured for 3 days under continuous longitudinal stretch in the presence and absence of PF‐4594755 , 2‐APB (30 μmol/L), and nifedipine (1 μmol/L) on PYK2 phosphorylation (panel A) and SM22α synthesis (panel D) in portal venous strips cultured for 3 days in the absence or presence of longitudinal load. PF‐4594755 and 2‐APB reduced PYK2 phosphorylation in both unloaded and loaded strips, but loaded strips still showed higher PYK2 phosphorylation. However, nifedipine selectively reduced PYK2 phosphorylation in loaded strips. Similar effects were seen on SM22α synthesis. Overall protein synthesis was significantly increased by stretch and was reduced in both stretched and unstretched strips by PF‐4594755 and 2‐APB, but only in stretched strips by nifedipine (data not shown). Therefore, relative to overall protein synthesis, neither PF‐4594755 nor 2‐APB had any effect on SM22α synthesis, while nifedipine selectively reduced its stretch sensitivity. Taken together, the effects of PF‐4594755 on PYK2 phosphorylation and protein synthesis are similar to those of 2‐APB but differ from those of nifedipine. This is further supported by the evidence that the combined action of PF‐4594755 and 2‐APB did not differ from that of PF‐4594755 alone, while the combination of PF‐4594755 and nifedipine reduced PYK2 phosphorylation to the same low level in both loaded and unloaded strips , smooth muscle α‐actin (Acta2), α‐tropomyosin (Tpm1) or desmin (Des). In particular, the activation of gene expression by stretch was unaffected by the PYK2 inhibitor, as was the basal gene expression. This is similar to the lack of effect of 2‐APB on stretch‐activated smooth muscle gene expression but contrasts with the inhibition due to block of voltage‐dependent calcium channel activity , likely via ligand‐independent activation  eliminated the stretch sensitivity of PYK2 and Akt phosphorylation but did not affect their basal levels , smooth muscle α‐actin (Acta2) and α‐tropomyosin (Tpm1)  Fig. D–F.The effect of stretch on smooth muscle growth and differentiation likely involves both integrin activation and increase of intracellular calcium. Among possible mediators of the response, PYK2 is interesting because of its activation both by integrins and calcium, its association with Src, G‐protein, and MAPK signaling and its interaction with L‐type calcium channels  stretch activates ERK1/2 despite PYK2 inhibition. We have previously shown that activation of ERK1/2 by acute stretch in portal vein is insensitive to both verapamil and 2‐APB  channels has been shown to have a special role in stimulating smooth muscle differentiation  channels (Beech A 10‐min stretch was here shown to increase PYK2 phosphorylation in portal venous strips that had been equilibrated overnight without load. This increase was sensitive to PF‐4594755 whereas basal PYK2 phosphorylation was not affected, although the inhibitor had been present during the equilibration. After a 3‐day culture, however, PYK2 phosphorylation was found to be reduced in both loaded and unloaded strips cultured with the inhibitor. A possible explanation for this is that constitutive PYK2 phosphorylation is low in short‐term culture but increases with time, as was seen here to be the case in unloaded portal vein. It is notable that organ culture of vascular smooth muscle, including the portal vein, tends to increase the expression of non‐voltage‐dependent ion channels as well as intracellular calcium release on agonist stimulation (Dreja et al. ls Beech . These c1 receptor inhibition by losartan produced similar results as PF‐4594755 on PYK2 phosphorylation and gene expression. At the concentrations of PF‐4594755 used here, we did not find significant increase of smooth muscle marker genes. However, an indirect indication that PYK2 inhibition may in fact contribute to maintenance of the smooth muscle in a contractile phenotype is the higher level of spontaneous activity noted after culture with the inhibitor, which may indicate inhibition of phenotype shift during culture. The mechanism behind this effect is not revealed by the present results, but it may be speculated that the increase in basal PYK2 phosphorylation with time in culture may correlate with a phenotype shift involving altered ion channel activity.Since PYK2 has been shown to mediate calcium‐dependent RhoA activation in response to Ang II in vascular smooth muscle cells (Ying et al. Interestingly, Zhang et al.  observedμmol/L PF‐4594755 was found to inhibit PYK2 to 96% and FAK to 42% (Bonnette et al. Some limitations of this study in relation to its possible pathophysiological relevance need to be addressed. The portal vein is unique in being a large vessel with phasic myogenic activity, which in some respects resembles that of myogenically active microvessels, and it is also highly responsive to increased transmural pressure (Albinsson et al. In conclusion, we have shown that PYK2 signaling functionally distinguishes effects of voltage‐dependent and non‐voltage‐dependent calcium influx on stretch‐sensitive smooth muscle growth and differentiation. PYK2 may be interesting for specifically targeting growth processes in phenotypically modified relative to contractile vascular smooth muscle.We thank Pfizer Inc. for the gift of PF‐4594755.Leonard Buckbinder is an employee of Pfizer, Inc. All other authors declare no conflict of interest.


Carcinus maenas), a species highly variable in appearance and found in a wide range of habitats. We used field surveys and digital image analysis of the colors and patterns of crabs found in four locations around Cornwall in the UK to quantify how individuals vary with habitat . We find that individuals from sites comprising different backgrounds show substantial differences in several aspects of color and pattern, and that this is also dependent on life stage (adult or juvenile). Furthermore, the level of individual variation is dependent on site and life stage, with juvenile crabs often more variable than adults, and individuals from more homogenous habitats less diverse. Ours is the most comprehensive study to date exploring phenotype-environment associations for camouflage and individual variation in a species, and we discuss the implications of our results in terms of the mechanisms and selection pressures that may drive this.Camouflage is widespread throughout the natural world and conceals animals from predators in a vast range of habitats. Because successful camouflage usually involves matching aspects of the background environment, species and populations should evolve appearances tuned to their local habitat, termed phenotype-environment associations. However, although this has been studied in various species, little work has objectively quantified the appearances of camouflaged animals from different habitats, or related this to factors such as ontogeny and individual variation. Here, we tested for phenotype-environment associations in the common shore crab ( Animals show substantial variation in appearance among, and sometimes within species. The selection pressures underlying and driving this are varied, with multiple mechanisms often operating in the same population Jaculus jaculus) over large geographical scales, most likely driven by selection for camouflage from predators. This work showed correlations between the colors of individuals and predicted colors of the background from satellite images for where individuals were collected Despite the above comparatively simple predictions, there remain substantial gaps regarding research on phenotype-environment associations and camouflage. Work to date has been largely restricted to investigating broad changes in overall body coloration without rigorous analyses of individual appearance, and has not investigated species with more complex patterns. For example, a recent study showed phenotype-environment associations in African desert jerboas , each associated with utilizing different substrates in their rocky shore habitat Phenotype-environment associations for camouflage are likely to occur frequently in invertebrates too, including commonly in marine environments. For example, the isopod Carcinus maenas) in different habitats. Powell 2), meso (100 s m2), and macro , and found evidence for associations between proportion of patterned and unpatterned crabs at each scale with different substrate types . The strongest associations were at the micro scale, with higher proportions of patterned crabs on mussel beds than on rocks or algae.Perhaps the most in depth assessment of phenotype-environment associations for camouflage so far has been conducted in shore crabs , and the level of inter-individual variation in appearance and how this relates to habitat. To human eyes, crabs are often extremely well camouflaged , and theShore crabs are an ideal species to address these issues, being the most widespread crab species in the UK and much of Europe, and found in a wide range of habitats  each with different appearances We used four locations to sample crabs from around the Cornish peninsular in the southwest UK, which differed in the predominant habitat type. Site 1 was Falmouth  on the south coast, comprising a stretch of shoreline collectively encompassing Castle and Gyllyngvase beaches. The site primarily comprises large areas of rockpools and rocky crevices with stony/gravel substrates in the pools, and lower down the rockpools increasing amounts of seaweed. Site 2 was Godrevy Point  on the north coast, which primarily consisted of exposed rocky outcrops with mussel beds. Site 3 was Helford Passage, an estuarine location on the south coast  comprising a large mudflat area, as well as some tiered craggy rock pools. Finally, site 4 was Summers beach at St Mawes , also on the south coast, and comprising rockpools, gravel, and some low seaweed cover adjacent to a pebbled beach.We conducted sampling at each site over three broad time periods to be sure that our sampling was not restricted to one interval that may be affected by changes in crab abundance or distribution . Sampling was conducted in 2013 with periods broadly being mid April to early May, late May to early June, and mid August to early September, although owing to weather and tides some sampling days were somewhat later. We visited each site three times on different dates, roughly separated into spring, early summer and late summer. For each site and for any given sampling period we conducted one transect at three different heights on the shore . Originally, we split Falmouth into two areas (Gyllyngvase and Castle) as these are separated by a small sandy beach, and therefore undertook three periods of sampling at each place. However, this area essentially forms one almost continuous area of rockpools along a small stretch of coastline, and so we decided it was more appropriate to pool this dataset into one location. As such, we undertook twice as many transects at Falmouth than for the other sites, and partly for this reason this area has a larger sample size than the other sites. All work was conducted under approval from the University of Exeter Biosciences ethics committee (application 2013/75). The field locations (specified above) are publically accessible land and no further licenses or permits were needed. Crabs were kept no longer than 3 hours, and all individuals were returned unharmed to their original rockpool area after being tested. Shore crabs are not an endangered or protected species.On each sampling event a 50 m (30 m at Godrevy point due to the nature of the beach) transect was placed horizontal to the shoreline corresponding to low, middle and high tide zones spaced evenly down the beach. Transect positioning was determined by measuring the depth of the beach and dividing into four sections. The upper transect was positioned approximately one quarter of the distance from the top of the mature rockpool area and the lower transect one quarter of the distance from low tide, with the middle transect positioned central to this. The approach was taken to ensure sampling on different heights of the beach while avoiding extreme low and high tide areas. Each transect was subdivided into 2.5 m intervals, whereby at each point a 0.5 m by 0.5 m quadrat was placed on either side of the transect. Each quadrat was then searched for shore crabs for a time period of five minutes . During the search, movable boulders, rocks and seaweed were turned over, crevices checked, and sand, mud and gravel raked using fingers in order to ensure crabs were unlikely to be missed.After collection crabs were placed in buckets (white for sample period 1 and grey sample periods 2 and 3). Crabs were sexed according to their abdomen Although the field locations were quite different in appearance to us, we sought to quantify the proportion of different substrate types found in each site. To do so, we ran a further nine transects of 30 m at each site (three at each shore height). At each 1 m interval we simply recorded the substrate type present. From this, for each site and shore height we calculated the proportion of each background type present, allowing us to determine the difference in habitat composition between locations and to relate this to differences in crab appearance. We undertook these transects in May 2014. Although this was not the same year as when the crab sampling took place, we could detect no major changes in the appearance or composition of the habitats between years, meaning that any error is unlikely to be substantial.Photography and initial image calibration and analysis generally followed our previous recent methods During calibration (undertaken in custom programs in Image J), images were converted to uncompressed TIFF files and the images of each crab comprised four bandpass layers corresponding to the long-wavelength (LW), medium-wavelength (MW), short-wavelength (SW), and UV parts of the spectrum. Most cameras show nonlinear responses in image value to changes in light levels that need to be corrected before accurate data can be obtained, and so we linearized all images based on quantified camera responses to a set of eight Spectralon grey standards with reflectance values ranging from 2–99% 2 = 0.98) between crab brightness values based on mediumwave only and overall reflectance based metrics  of the entire crab carapace pattern, but avoiding any clear areas of specular reflectance. We then calculated several metrics of appearance based on color, brightness, and pattern. Brightness was calculated as (LW+MW+SW+UV)/4, and is a measure of how bright or dark crabs are across the entire spectrum see also .a priori reason to define a specific color channel, we followed earlier approaches in using a principal component analysis (PCA) to determine the main axis of color variation that exists in the crabs, and to use this to inform our decision about a logical color channel(s) to use Next, we calculated two types of color metric for each crab, being saturation and hue. Saturation, generally considered as the amount of a given color compared to white light (i.e. how rich or deep the color is), can be calculated by plotting the position of each color in a tetrahedral space based on XYZ coordinates derived from the standardized  LW, MW, SW, and UV values Shore crabs also differ greatly in pattern as well as color, and so we quantified the key features of crab patterns using a ‘granularity’ analysis approach. This has previously been used to analyze both the camouflage of cuttlefish patterns e.g. , and birFor each granularity band, we calculated the overall pattern ‘energy’, being the sum of the squared pixel values in each image divided by the total number of pixels Therefore, overall we calculated six different measures of crab appearance from our image analysis techniques: brightness , saturation , hue , proportion energy , total energy , and marking size (the dominant marking size on the crab pattern).We also quantified the amount of variation that exists in both adult and juvenile crabs at each location. To do so, we utilized an approach previously developed to quantify polymorphism in the egg colors and patterns of parasitic and host birds in a ‘multidimensional phenotypic space’ (MDPS) In order to compare adult and juvenile crabs, we needed to be able to classify crabs into one age group or another. Sexual maturity in crabs can vary with size and location, and so initially we used two criteria to classify individuals based on previous work and our own initial studies, whereby the point of maturity is determined in relation to size. First, crabs were separated based on estimates of maturity taken in males and females We first used DFA to determine how effectively crabs were separated into our two potential age categories based on our appearance metrics (see above). For this we used the ‘lda’ function from the ‘MASS’ library in R To determine how crabs differ in appearance with location, we ran General Linear Models (GLMs) on each appearance trait  as the response, with factors site (field location), shore height , and life stage (adult or juvenile) as fixed effects. We also included interactions between site and height (to determine if changes in appearance with site depend on location on the shore), site and life stage (to determine if differences among site depend on adult or juvenile crabs), and height and life stage. Any non-significant interactions were removed from the model in a stepwise manner and the model rerun. Data generally needed transformation  including our appearance metrics defined above, to determine the level of overlap in crab appearances between each site and the proportion of crabs misclassified into the wrong site category. As above, we used a leave-one-out cross-validation DFA technique for this.Finally, for the MDPS variability data, we ran a GLM with MDPS values as the response, with site, life stage, and an interaction between site and life stage included in the model to test whether crabs from some locations are more variable than crabs from other sites, and whether this depends on age class (adult or juvenile).We sampled 677 crabs in total from four sites of different habitat composition (see below): 264 from Falmouth , 92 from Godrevy , 158 from Helford , and 163 from St Mawes .We used discriminant function analyses (DFA) to determine how effectively different life stages fall into predefined age groups, using the predictors of brightness, saturation, hue, total energy, proportion energy, and marking size. First, breaking down crabs into mature (n = 188) and immature (n = 489) for males and females, the proportion of correct classifications was to 0.790. Here, mature individuals are correctly assigned at a proportion of 0.423 , whereas immature crabs are correctly classified at a proportion of 0.930 . Second, breaking down crabs into juveniles (508) as individuals with carapace widths of <25 mm and adults (169) as crabs larger than this, produced a proportion of 0.836 crabs correctly assigned to their group. Adult crabs were correctly assigned at a proportion of 0.490 , and juveniles were correctly classified at a proportion of 0.953 (24 misclassified as adults). Therefore, the second analysis best separates crabs into adults and juveniles with least overlap between categories, and so we used these categories in the further analyses.s = −0. 496, t = −14.84, df = 675, p<0.001), and have less saturated colors . Larger crabs also have less contrasting carapace patterns , though unsurprisingly bigger crabs have larger markings . In addition, crabs have higher hue values as they get bigger . Here, crabs are moving closer to hue values of 1.00 as they get larger, essentially moving towards the achromatic point (i.e. becoming more grey/less blue-green in color). Note also that as crab sizes get larger the spread of values decreases, reflecting the fact that juvenile crabs show greater variation in appearance. Finally, crabs have lower proportion energy values when they are bigger, implying a larger range of marking sizes and less prominent markings .There were significant relationships with all appearance metrics . Larger There were clear differences in the substrate composition of each of our four sites . For FalIn contrast to Falmouth and St Mawes, Godrevy is a site dominated by mussel beds and a rocky substrate. There are few changes with shore height in substrate proportions. Helford, a site we have classified as mainly mudflats, shows clear changes in substrate type with shore height. At upper shore heights, the site is mainly composed of rocky substrate and gravel. Moving down the shore the substrate changes to become dominated by mud/sediment, with some green/brown algae and gravel. Note, however, that despite the predominantly rockpool nature of the upper shore, these rockpools contained large amounts of mud and sediment, such that the appearance of the background at all three shore heights was relatively similar (muddy).  = 3.11, p = 0.026), life stage , but not zone . Generally, juveniles have higher brightness values than adults, but the magnitude of this difference depends on the site, with particularly large differences in brightness between adults and juveniles at Falmouth and Godrevy. In general, crabs from Helford were darker, and with small differences between adults and juveniles. Interestingly, crabs from Falmouth are brighter lower down the shore, whereas crabs from St Mawes become darker  and there were significant effects in the model of site , life stage , but not zone . There were no significant interactions. Adults have lower saturation values than juveniles. Crabs from Helford are generally more saturated than crabs from other sites, though juveniles from St Mawes also show high saturation values. Adult crabs from Falmouth and Godrevy have lowest saturation values.For hue there were significant effects in the model of site , life stage , but not zone . There were no significant interactions. Crabs from Helford have the lowest hue values, which equates to more blue-green coloration. Crabs from other sites have coloration closer to grey (more red-brown), but note that much of the color differences would also occur in the color channel hue 1, which we did not analyze further due to the correlation with saturation see .There were substantial differences in pattern among crabs, demonstrated by the diversity of granularity spectra obtained . Data foFor total energy, the data was log transformed. There was a significant effect in the model for only site , but not life stage , or zone . There were no significant interactions. Crabs from St Mawes had higher total energy values (more contrasting markings), whereas crabs from Godrevy and Helford had relatively low values.Marking size data was Box Cox transformed (power of 0.113). There was a significant effect in the model of life stage , but not zone . There was only a non-significant trend for differences among sites . Here, larger crabs had bigger markings, as would be expected with the increase in body size.We tested whether there was a difference in the size of crabs among sites and between the three heights on the beach with a GLM (data log transformed), with carapace width as the response and site and zone in the model. There was no difference between the size of crabs for either site , or zone , nor was there a significant interaction between site and zone.We conducted DFA on the appearances of crabs among sites for both adults and juveniles based on the metrics of color and pattern . JuvenilWe compared whether crabs from different sites are more or less variable in a multidimensional phenotypic space (MDPS), and this revealed some clear differences in the individual diversity of crabs from different sites and age categories . The datHere, we tested for phenotype-environment associations in the shore crab among four sites of different habitat composition, and for differences between adults and juveniles. First, we found significant differences in the appearance of crabs among each site for color, brightness and pattern. Adult and juvenile crabs showed clear differences, with adult crabs generally darker and less saturated in coloration than juveniles. Adults also had less contrasting or prominent markings, and larger making sizes. However, these differences sometimes depended on the site. We also found corresponding changes in appearance associated with continuous increase in crab size. Furthermore, as crabs get larger the spread of values in the correlations decreases for many of these appearance metrics, reflecting the fact that juvenile crabs show greater variation. These differences with size/age also translate into a high level of distinctiveness between adult and juvenile crabs. Our discriminant function analysis (DFA) showed that approximately 84% of crabs are correctly classified as either adult or juvenile based on our appearance metrics. Differences in appearance between adult and juvenile crabs, and specifically less diversity in adult crabs, have been widely reported before e.g. , but ourRegarding differences among sites, crabs from Helford, a site characterized by relatively dark green-brown mudflats and sediment, were generally darker than crabs from other sites. There was little change in brightness with zonation, perhaps because this habitat varies less in appearance overall, which is consistent with our observations that even the rockpools on the upper shore contained a great deal of mud, even though the background types did vary with zone. Crabs had lower hue values at Helford than elsewhere, meaning that they were more blue-green in coloration, and they had relatively high saturation values meaning that their color was richer or less achromatic. Helford crabs also tended to have relatively low total energy values, illustrating that their patterns were not very prominent, again consistent with the more homogeneous mud background. In contrast, crabs from Falmouth (mainly rockpools) are brighter lower down the shore than higher up. This may be because higher up the shore has more dark rock, whereas lower down the shore there is a greater amount of green and brown algae and other changes in substrate, which may be brighter in coloration or allow different types of camouflage. There are also considerable differences in the brightness of adults and juveniles, with juveniles having higher reflectance and also being brighter than crabs from other sites. Both adult and juvenile crabs from Falmouth have relatively high proportion energy and total energy values, indicating that they have relatively prominent markings. This is in accordance with the relatively high-contrast backgrounds found at this site, with lots of rocks and substrate of different colors and of varied grain size, and the wide variety of background substrate types . Crabs fWe also used DFA to determine how distinct crabs from each site are in overall appearance. The results showed that for both juveniles and adults, 43–53% of individuals are correctly classified to their respective sites. Although this is not especially high , given that there were four sites studied and that the sites overlap to a lesser or greater extent in habitat substrate , this leIn terms of the level of individual variation, crabs from St Mawes (both adults and juveniles) were highly variable in the multidimensional phenotypic space (MDPS). This is likely to stem from the (subjectively) considerable variation in background appearances at this location, although the site did not have a large range of substrate types present. In contrast, crabs from Godrevy and Helford, as well as adult crabs from Falmouth, had relatively low MDPS values. For Godrevy and Helford, this makes sense because the mussel bed and mudflat backgrounds at these locations are relatively uniform in appearance . At Falmouth, juvenile crabs, however, were highly variable. This difference is intriguing and we think it most likely reflects differences in matching to the substrate scale with size (see below), perhaps also linked to the wide range of substrate types present.2) scale, something which we did not investigate here, and this would be important to further investigate in future work.Overall, we have shown clear differences in the appearance of crabs among sites. In addition, we also show how variation in appearance among sites is often quite different between adults and juveniles, and find clear differences in the amount of individual diversity associated with site. While other studies have investigated phenotype-environment associations in shore crabs before Pollachius pollacbiusScyliorhinus canicula), which appear to be monocromats Our results demonstrate clear differences in crab appearance associated with site/habitat. It should be noted, however, that as with past work we have not shown a direct link to camouflage. This requires demonstrating that differences in the appearance of crabs among sites translates into improved camouflage at their own location versus other locations; that is, phenotype-environment matching. It will be important to do this in future work. However, this is non-trivial for several reasons. First, the range of backgrounds encountered, with many being underwater or partially submerged, makes quantifying the background challenging. We were unable to accurately quantify background appearances in the current work, although this is a major future aim of ours. Second, camouflage needs to be analyzed to predator vision, yet crabs are attacked by a wide range of species, especially various bird and fish Regardless of the lack of direct quantification of camouflage , we believe that camouflage is by far the most likely explanation for our results. First, crab differences in appearance with habitat (and for brightness with shore height too) cannot stem from differences in size since we found no difference in the sizes of crabs among sites/zones. In addition, as Todd et al. 2 scale) is likely to be driven by differential predation on mismatched crabs, short term color change, and behavioral choice of substrates, at the meso-scale and medium time periods (ca. weeks and months and 100 s–1000 s m2) by longer term color change and phenotypic plasticity through molting, and at the macro-scale and over longer term  by differential predation and genetic adaptation.The presence of phenotype-environment associations in this and previous work raises the question of what mechanisms drive it. As Todd et al. Like many other animals Variation and matching among sites could also arise if populations evolve genetic differences, and there is some evidence for distinct genetic populations of shore crabs across large geographic regions of Europe Lottia digitalis has camouflaged shell colors that vary in appearance. Byers Finally, it is possible that crabs are able to behaviorally choose backgrounds that improve their camouflage. Indeed, we have found some preliminary evidence that adult crabs at least will choose between substrates of different appearance (unpublished data). This mechanism may underlie associations at the micro scale, such as with different shore heights. Camouflage based on behavioral choice is likely to be more common than previously thought across a wide range of species, and there is growing evidence that some animals select backgrounds and resting postures to improve their match to the background, including some birds and moths e.g. . BehavioThe question of why crabs change appearance as they grow and become less patterned with age is somewhat of a mystery, although there is range of potential explanations, many of which are not mutually exclusive. First, smaller shore crabs may be associated with different substrate types Phenotype environment matching and camouflage has the potential to help us to understand how animals adapt to different environments and the selection pressures that produce this. In addition, the subject can be a valuable means to explore the mechanisms that produce environment associations, and what drives individual variation and diversity in general, and their relative importance. Crabs are an especially valuable group to utilize because they often suffer considerable predation pressure and show a wide range of adaptations for camouflage S1 DataExperimental data.(XLSX)Click here for additional data file.


A specific issue was to determine the relative role of phasic and tonic inhibition in dynamically regulating granule cell firing, which has not been clarified yet. The model comprised the excitatory mossy fiber—granule cell and the inhibitory Golgi cell—granule cell synapses and accounted for vesicular release processes, neurotransmitter diffusion and activation of different receptor subtypes. Phasic inhibition was based on stochastic GABA release and spillover causing activation of two major classes of postsynaptic receptors, α1 and α6, while tonic inhibition was based on steady regulation of a Cl− leakage. The glomerular microcircuit model was validated against experimental responses to mossy fiber bursts while metabotropic receptors were blocked. Simulations showed that phasic inhibition controlled the number of spikes during burst transmission but predicted that it specifically controlled time-related parameters (firing initiation and conclusion and first spike precision) when the relative phase of excitation and inhibition was changed. In all conditions, the overall impact of α6 was larger than that of α1 subunit-containing receptors. However, α1 receptors controlled granule cell responses in a narrow ±10 ms band while α6 receptors showed broader ±50 ms tuning. Tonic inhibition biased these effects without changing their nature substantially. These simulations imply that phasic inhibitory mechanisms can dynamically regulate output spike patterns, as well as calcium influx and NMDA currents, at the mossy fiber—granule cell relay of cerebellum without the intervention of tonic inhibition.The complex interplay of multiple molecular mechanisms taking part to synaptic integration is hard to disentangle experimentally. Therefore, we developed a biologically realistic computational model based on the rich set of data characterizing the  Synaptic inhibition controls local microcircuit functions by regulating membrane potential and calcium influx in neurons, with important consequences on spike generation and synaptic plasticity . The patch-clamp pipette solution contained (in mM): 81 Cs2SO4, 4 NaCl, 2 MgSO4, 0.02 CaCl2, 0.1 BAPTA, 15 glucose, 3 ATP-Mg, 0.1 GTP, and 15 HEPES. For experiments with current-clamp recording the internal solution contained (in mM): 126 potassium gluconate, 4 NaCl, 5 Hepes, 15 glucose, 1 MgSO4, 0.1 BAPTA-free, 0.05 BAPTA-Ca, 3 Mg2+-ATP, 0.1 Na+-GTP. These solutions maintained resting free [Ca2+] at 100 nM and pH was adjusted to 7.2. Patch-clamp pipettes filled with these solutions had a resistance of 5–8 MΩ before seal formation. Transient current analysis yielded a membrane capacitance (Cm) of 3.9 ± 0.3 pF, membrane resistance (Rm) of 2.0 ± 0.2 MΩ, and series resistance (Rs) of 17.1 ± 0.9 MΩ (n = 7). The 3-dB cell plus electrode cut-off frequency was fVC = (2π RsCm)−1 = 2.5 ± 0.1 kHz (n = 7). All drugs were obtained from Sigma, except BAPTA tetrapotassium salt ; GABA-B receptor blockers CGP35348 and CGP55845, mGluRs blockers MCPG, CPPG and gabazine . All experiments were conducted in accordance with international guidelines from the European Community Council Directive 86/609/EEC on the ethical use of animals.Patch-clamp recordings in acute cerebellar slices were performed as reported previously  after gabazine perfusion (on average 21.50 ± 2.12 pA n = 7) and sfEPSC amplitude = −25.8 ± 3.3 pA, the EPSC/sfEPSC amplitude ratio yielded an average of 3.14 activated fibers per granule cell. The number of activated Golgi cell axons was estimated by the ratio between peak amplitude of evoked IPSCs (eIPSC) and spontaneous IPSCs (sIPSC) in the same granule cells. The sIPSCs depend on Golgi cell autorhythmic activity and are generated at single connections  recorded from granule cells in similar experimental conditions  or indirectly on extrasynaptic receptors through spillover from neighboring sites (slow indirect component)  that well reproduces synaptic dynamics on short time scale, as already demonstrated at the mossy-fiber to granule cell synapse , the diffusion coefficient of GABA was set equal to that of glutamate . Then, the whole eIPSC train was fitted on data from our previous experimental investigation  and release probability (p).Stochastic model. In order to test the GRC responsiveness in a more natural context we introduced synaptic stochastic mechanisms at the excitatory and inhibitory synapses. We modeled stochastic pre-synaptic release of the excitatory and inhibitory phasic components . The stochastic quantal release model was implemented as in Arleo et al. (rnd) is generated at each RS where a fixed concentration of neurotransmitter (Tmax) is released when the corresponding rnd falls below the actual releasable resources (Y). This stochastic model yields the deterministic response when averaged over multiple trials (not shown).o et al.  by splitIn order to test the responsiveness of the granule cell (GC) in a realistic context, a GC microcircuit was built that included the model of the GC . The results obtained with the second protocol are reported in Figures 7, 8.Two stimulation protocols were used to simulate the impact of the different inhibitory mechanisms on the granule cell output. First, a high frequency input  was used to activate the excitatory synapses, while the inhibitory synapses were activated by the same input train delayed by 5 ms to mimic synaptic and integration delays in the feed-forward inhibitory MF-GoC-GC pathway. The results obtained with the first protocol are reported in sn), the timing of the first spike (ttfs), the first spike standard deviation  and the time window of the output spikes . Reliable estimates of the measured parameters were then obtained by running 500 runs for each tested condition.To the ease of comparison with experimental results we monitored set of parameters such as the number of output spikes (www.python.com) based on the Scipy (www.scipy.com), Numpy (www.numpy.com) and MatPlotLib (www.matplotlib.com) modules. The analysis allowed to quantify the number of spikes elicited, the time window, the time of the first spike and its variability  in the tested E/I conditions. In a set of simulations we also recorded the Calcium and NMDA current traces and computed the mean values over a 130 ms long time window.All data were analyzed with custom codes developed in Python  was obtained by reporting the excitatory and inhibitory strengths on Cartesian coordinates using a color-code map, in which a color scale corresponded to the intensity of the measured parameter. The excitatory, inhibitory strengths  were quantified as the sum of the active fibers (MF/GoC) and their release probability (Pe/Pi), such that ES = MF + Pe and IS = GoC + Pi.A compact representation of the E/IS was calculated on sub-regions of the E/I space by averaging over the active fibers involved and the corresponding release probabilities . Each sub-region was named after the number of active fibers involved: for example, E/I(1/1) corresponds to 1 excitatory and 1 inhibitory fiber. E/IS yields a comprehensive quantification of the changes of the measured quantities at variable E/I levels.The E/IS data distributions at variable phase-lag between synaptic excitation and inhibition, the data were fitted with a Lorentz equation y(θ) of the form /2. Lorentzian fittings allowed to find the resonance phases (RF = maxθ) and to calculate RA = maxy(θ)/ω, which corresponds to the quality factor Q in resonance literature and characterizes a resonator's bandwidth relative to its center delay. The goodness of fit was assessed by calculating the squared correlation factor, R2. Q is normally used in signal processing theory and describes the tendency of a signal to show resonance vs. monotonic roll-off.In order to describe the shape of E/I response space representation also distinguishes between different settings of the GABA tonic current when the phasic component is zero.The sn, tw, ttfs) on the E/I space we calculated the coding fraction  is the entropy of the E/I input space and MI quantifies the mutual information of the measured parameters P . Each E/IS configuration of the E/I space was regarded as an independent stimulus. The cardinality of the stimuli is therefore given by: 4 (MF) × 9 (Pe) × 5 (GoC) × 9 (Pi) = 1620. PCF measures the fraction of E/IS that can be distinguished based on the observation of the whole output generated by the model  and is normalized between 0  and 1 . CF calculation was performed with the Python module “pyentropy” , the discharge time-window increased  while the time-to-first-spike and first spike standard deviation did not vary significantly  . Since metabotropic mechanisms can influence granule cell responses  Figure . These rp < 0.05, n = 7) and time window  but did not significantly change the time to first spike  or first spike standard deviation   Figure . Thus, aIn each of the recorded granule cells, measurements of EPSCs and IPSCs were obtained at the reversal potential of IPSCs and EPSCs, respectively Figure . MoreoveFigure 6).The number of active synapses was estimated by comparing EPSCs and IPSCs with the average single-site responses measured previously in identical experimental conditions Figure  Sola et. The estA model of cerebellar glomerular inhibition should be able to predict the different kinds of synaptic inhibitory responses observed experimentally in granule cells.mIPSCs, which correspond to release of single neurotransmitter quanta : (i) the vesicular release cycle contained an explicit representation of the number of releasing sites and of release probability, (ii) the mechanism of vesicle fusion was stochastic, (iii) the postsynaptic membrane expressed α1 and α6 receptors, (iv) GABA diffusion was explicitly modeled, and (v) tonic GABA receptor activation was accounted for.1-type; Brickley et al., 6-type; Tia et al., eIPSCs were reconstructed as the sum of α1 and α6 receptor-mediated components , in close agreement with experimental determinations . The normalized eIPSC amplitude showed a significant difference on the first pulse but then recovered to control values and the total electrical charge transferred during the IPSC train was in good agreement with the experimental one and did not change significantly by raising pI   Figure . These sdendritic microcircuit model  synapse combinations and release probabilities was performed generating multiparametric color plots  from a high response region . Moreover, a micro-pattern emerged inside each sub-quadrant determined at each E/I by varying excitatory and inhibitory release probabilities, pE/pI. Thus, in principle, by combining different numbers of fibers with different release probabilities, a broad set of response combinations could be obtained generating an extended response space. A quantitative insight on its properties was obtained by considering representative areas. For example, by comparing parameters in the quadrants E1,2/I3,4 (low E/I) and E3,4/I1,2 (high E/I) there was a nearly 10-fold increase in number of spikes, a 3-fold increase in time window and a modest changes in time to first spike and first spike standard deviation  of GABAergic inhibition  of number of spikes, time window, time to first spike and first spike standard deviation. Conversely, raising pE remarkably increased number of spikes (≤80%) and decreased time to first spike and first spike standard deviation (≤80%) with a limited effect on time window (≤20%)  Figure . TherefopE/pI intersection . It should be noted that this data distribution was not significantly different from the unitary diagonal (X2p < 2.6 e−10). Finally, it should be noted that time to first spike and first spike standard deviation showed modest changes compared to number of spikes and time window, in agreement with experimental results matched for the E/I balance . Therefore, the model was able to capture the main quantitative properties of glomerular transmission and the impact of synaptic inhibition on granule cell firing.The simulated parameters were compared to those measured experimentally , the coding fraction  was calculated . The most apparent changes occurred when α6 receptors were switched off. Interestingly, number of spikes, time window, time to first spike and first spike standard deviation showed resonance around −10 ms phase-lag. When α1 receptors were switched off, resonance appeared around 10 ms phase-lag. The impact of the E/I phase was evaluated also for currents generated by NMDA channels and voltage-dependent calcium channels (VDCCs)  Figure . Again, These simulations showed that α1 and α6 receptor-mediated effects combined in tuning spike generation toward specific phase-lags and that α6 receptor-mediated effects were larger and broader than α1 receptor-mediated effects.E/I phase-lag. This effect was evaluated by fitting the tuning curves with Lorentzian functions yielding Q = A/ω, a parameter indicating the tendency of the response to peak at a certain phase-lag maxθ with area A and broadness ω  > Q(α1) reflected the larger impact of α6 than α1 receptor-mediated charge transfer, ω(α6) > ω(α1) reflected the longer duration of α6 than α1 receptor-mediated responses, and maxθ(α6) < 0 and maxθ(α1) > 0 reflected the slower raise of α6 than α1 receptor-mediated responses  was much larger for time window, time to first spike and first spike standard deviation than for number of spikes  of the NMDA current was very small, reflecting the slow time course of this current and its broad integration times  for time window, time to first spike and first spike standard deviation tended to decrease with E/I, according to the powerful effect of phasic inhibition in controlling time-dependent parameters  with E/I for the VDCC current followed strictly that of the number of spikes  with E/I for the NMDA current was marginal but, specifically, maxθ (α6) shifted progressively from −20 to +30 ms  when the E/I phase was changed. In all conditions, the overall impact of α6 was larger than that of α1 subunit-containing receptors. However, α1 receptors controlled granule cell responses in a narrow ±10 ms band while α6 receptors showed broader ±50 ms tuning. Eventually, phasic inhibition fine-tuned the initiation and termination of granule cell firing during burst-burst transmission, as well as the probability of firing at different E/I phases, on the millisecond-scale. The model also predicts that tonic inhibition can bias these effects without changing their nature substantially. These results imply that the specific organization of phasic inhibitory mechanisms can effectively regulate the dynamics of spike generation at the mossy fiber - granule cell relay of cerebellum without the intervention of tonic inhibition.In this paper we have developed a detailed glomerular microcircuit model could fit and explain the firing patterns observed in experimental data following mossy fiber burst stimulation. Once experimental granule cell firing parameter  values were reported on the simulated response space, the glomerular microcircuit model yielded pE and pI values corresponding to those observed experimentally  but had minor effects on time-related parameters, time to first spike and first spike standard deviation. The pE/pI balance was most effective in controlling number of spikes, time to first spike and first spike standard deviation. Raising pE specifically increased number of spikes and reduced time to first spike, consistent with the fact that AMPA and NMDA receptor activation shows a remarkable modulation with pE  showed much sharper tuning than tonic firing (number of spikes). Moreover, tuning of time-related parameters decreased with E/I (from −20 to +30 ms phase-lag) suggesting a complex voltage-dependent interaction between NMDA channel unblock, depolarization and inhibitory control.The model also predicted how VDCC and NMDA currents would be regulated by α1 and α6 receptors. VDCC current regulation strictly followed the number of spikes, as these channels open during the upstroke . Therefore, in summary, the α6 component dominated on feed-forward inhibition, while the α1 component gained relevance in controlling the timing of granule cell output firing at low E/I during feed-back inhibition. Interestingly, during feedback inhibition at low E/I the α1 component is often more effective than the α6 component. Concerning the NMDA current, phase-lag precession suggested that the maximum impact of α6 mediated inhibition at 5–10 ms phase-lag was also achieved at low E/I. The multiple combinations of these effects and the complex inhibitory patterns provided by α1 and α6 subunit-containing GABA-A receptors make phasic inhibition particularly suitable to finely regulate the delay and precision of granule cell firing.The dynamic control of inhibition by α6 and α1 receptors could have specific consequences during local microcircuit activation. These properties may have an impact on spike-timing dependent plasticity (STDP) induction and on its interaction with low-frequency coherent oscillations reported in the cerebellar granular layer   ratio. Since short bursts  without affecting time-related parameters (time to first spike and time window) significantly. (2) During inhibition with variable E/I phase, the pattern observed during bursts is reversed, so that phasic inhibition specifically affects time-related parameters without affecting tonic spike discharge significantly. (3) During inhibition with variable E/I phase, tonic GABA leakage does not alter the dynamics of inhibition substantially, so that tonic inhibition simply scales the effect of phasic inhibition on firing parameters. (4) The differential kinetics imposed by α1 and α6 receptors during inhibition with bursts or with variable E/I phase may not be explained by tonic inhibitory effects. (5) Phasic inhibition reduced redundancy specifically for time-related parameters.In summary, simulations identify five main reasons why phasic inhibition is needed for dynamic control of spike transmission at the mossy fiber—granule cell relay. (1) During burst transmission, tonic inhibition simply scales the effect of phasic inhibition on tonic spike discharge (Biologically realistic simulations show that phasic inhibition, by exploiting the different kinetics of α1 and α6 receptor-mediated responses, can effectively regulate the delay and precision of granule cell firing. The effect of α6 receptors dominated during burst transmission while that of α1 receptors emerged during single-pulse transmission with variable phase lag, so that α1 and α6 receptors proved suitable for single-spike and burst control, respectively. These mechanisms in turn may regulate coincidence detection of calcium signals required for STDP and for its integration during theta-cycles (D'Angelo and De Zeeuw, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.