In patients with advanced non-small-cell lung cancer, vitamin D receptor (VDR) polymorphisms and haplotypes are reported to be associated with survival. We hypothesized that a similar association would be observed in patients with head and neck squamous-cell carcinoma (HNSCC).I C/T (rs10735810), BsmI A/G (rs1544410), ApaI G/T (rs7976091), and TaqI T/C (rs731236) were genotyped by sequencing in 204 consecutive patients with HNSCC who underwent tumor resection. Progression-free survival was compared between VDR polymorphisms using Kaplan-Meier survival curves with log-rank tests and Cox proportional hazard models adjusting for age, gender, smoking status, primary tumor sites, postoperative stages, existence of residual tumor, and postoperative treatment with chemotherapy or radiotherapy.In a post-hoc analysis of our previous prospective cohort study, VDR polymorphisms including Cdx2 G/A (rs11568820), FokI T/T genotype was associated with poor progression-free survival: median survival for T/T was 265 days compared with 1,127 days for C/C or C/T . In contrast, the other polymorphisms  showed no significant association with progression-free survival. The A-T-G (Cdx2-FokI-ApaI) haplotype demonstrated a significant association with a higher progression rate (P = 0.02).During a median follow-up of 1,047 days, tumor progression and death occurred in 76 (37.3%) and 27 (13.2%) patients, respectively. The FokThese results suggest that VDR polymorphisms and haplotypes may be associated with prognosis in patients with HNSCC, although the sample size is not large enough to draw definitive conclusions. Although a variety of VDR polymorphisms were reported to be associated with different disease phenotypes in previous studies, the functional effects of the VDR polymorphisms Cdx2 and FokThis was a post-hoc analysis of VDR polymorphisms in our prospective cohort study up to February 2008 In each case, tumor samples from the primary site and surrounding normal tissue, but not metastatic sites, were stored at −80°C after excision. Cancer tissue was divided into 2 specimens: 1 for pathological confirmation, where the sample was composed of more than 70% cancer cells, and the other for DNA extraction. Peripheral blood samples were collected during the preoperative period from the same patients to confirm whether alteration of the sequence was due to genomic polymorphisms or somatic mutations.I and Cdx2 were amplified by polymerase chain reaction (PCR) using the following primers (forward/reverse): for Fokl (rs10735810), ctccgaaggcactgtgctcaggcct/atggaaacaccttgcttcttctccctc; sequence, ggcctgggccctggggagat; parameters: denaturation at 98°C for 1 min, followed by 30 cycles at 98°C for 10 s, annealing at 68°C for 4 min, and the stopping reaction at 16°C. For Cdx2 (rs10735810), gggaaggagggagggaggaaggaagg/agctgtagcaatgaaagcaaacc; sequence, tagaaaacattgtagaacatc; parameters: denaturation at 95°C for 3 min, followed by 30 cycles at 95°C for 90 s, annealing at 59°C for 90 s, then at 72°C for 2 min, and 16°C. For BsmI (rs1544410), gctgagggccagctgggcaacctgaa/aaccagcgggaagaggtcaaggg; sequence, gggcaacctgaagggagacgtagc; parameters: denaturation at 94°C for 3 min, followed by 35 cycles at 94°C for 20 s, annealing at 62°C for 40 s, extension at 72°C for 1 min, then final extension at 72°C for 6 min, and 16°C. For ApaI (rs7976091) and TaqI (rs731236), agagcatggacagggagcaaggccaggcag/gcgcaggtcggctagcttctggatcatc: sequence, agagcatggacagggagcaaggccaggcag; parameters: denaturation at 94°C for 10 min, followed by 35 cycles at 93°C for 45 s, annealing at 66°C for 30 s, extension at 72°C 45 s, then final extension at 72°C for 10 min, and 16°C.DNA was extracted from the fresh frozen tumor and peripheral blood samples using QIAcube  following the manufacturer's protocol. DNA fragments including FokThe PCR products were incubated with Rapid alkaline phosphatase  and Exonuclease I  at 37°C for 30 min, then 80°C for 15 min. Using Big Dye Terminator v3.1 Cycle Sequencing kit , the aliquots were incubated under the following conditions: 96°C for 5 min; 25 cycles of 96°C for 10 s, 50°C for 5 s, 60°C for 2 min. Treated PCR products were sequenced with the ABI PRISM 3700 Genetic Analyzer . Sequencing was confirmed with independent duplicate analyses.Mann-Whitney test and chi-square test were used to evaluate differences in patients' characteristics stratified by VDR genotypes. Progression-free survival was defined as the length of living time after surgery during which no apparent progression of cancer was found; curves were drawn using the Kaplan-Meier method and compared by VDR polymorphisms using log-rank tests. Cox proportional hazard models were fitted for multivariate analysis adjusting for age, gender, smoking status, primary tumor sites, postoperative stage (stage I to stage IV), existence of residual tumor, and postoperative treatment with chemotherapy or radiotherapy. Adjusted hazard ratios (HR) and 95% confidence intervals (CI) were computed. All statistical analyses were performed using STATA 12.0 . P<0.05 was considered statistically significant.Hardy-Weinberg equilibrium was assessed by the chi-square test. Linkage disequilibrium relationships of VDR SNPs that were not significant as assessed with Hardy-Weinberg equilibrium were visualized based on calculating D′. Genotype and allele frequencies were compared between progressed and non-progressed patients by means of chi-square analysis. Global differences in haplotype frequencies between progressed and non-progressed patients were assessed using 10,000 permutations for each comparison to correct for inaccurate p-values due to multiple comparisons. All analyses were done using Haploview Patients' age ranged from 32 to 89 years, and there were more men than women. More than half of patients (55%) were smokers; 64% of smokers were men. Most patients had oropharyngeal cancer, and 51% had stage IV disease. In 10 patients, tumor tissue was not resected completely. Thirty patients underwent chemotherapy or radiotherapy after surgery.I (P = 2.7×10−5) and TaqI (P = 0.04). Linkage disequilibrium analysis between VDR polymorphisms was shown (I and TaqI was 78. Patients' characteristics stratified by FokI genotype are shown in I or other polymorphisms (data not shown).We confirmed that polymorphisms were common between the tumor and peripheral blood samples obtained from the same patients. Genotype and allele frequencies of the VDR polymorphisms are shown in as shown . D′ betwP = 0.0004)  showed no significant associations with progression-free survival (data not shown).All 204 patients were followed for a median of 1,047 days , during which 76 (37.3%) progressed and 27 (13.2%) died of HNSCC. Kaplan-Meier survival curves showed that the FokI T/T genotype was associated with poor prognosis: median progression-free survival for T/T was 265 days compared with 1,127 days for C/C or C/T (log-rank test:  0.0004) . In contI with adjustment for age, gender, smoking status, primary tumor site, postoperative stage, existence of residual tumor, and postoperative treatment with chemotherapy or radiotherapy  showed no significant association with progression-free survival (data not shown).Cox proportional hazard models were computed to determine the significance of Fokotherapy . WithoutI and TaqI, only Cdx2, FokI, and ApaI were used in haplotype analyses. Associations between VDR haplotypes and disease progression were analyzed with permutation analysis (I-ApaI) haplotype demonstrated a significant association with a higher progression rate (P = 0.02).Because Hardy-Weinberg equilibrium test was significant for Bsmanalysis . The A-TI T/T genotype was associated with a poor progression-free survival rate in patients with HNSCC, even after adjusting for age, gender, smoking status, primary tumor site, cancer stage, residual tumor, and postoperative treatment (chemotherapy or radiotherapy). Arai et al. demonstrated that compared with the FokI T/T genotype, FokI C/C had 1.7-fold greater function of vitamin D-dependent transcriptional activation of a reporter construct under the control of a vitamin D response element in transfected HeLa cells I T) to the ACG (FokI C) polymorphism, the first potential start site moved to the 3′ direction, resulting in proteins that were 3 amino acids shorter and more functional I T/T may have less response to vitamin D, resulting in a higher progression rate.In this study, we found that the VDR FokI-ApaI) was associated with poor prognosis. This result is similar to previous studies showing that lower serum 25(OH)D levels and the Fokl T/T genotype as well as a VDR haplotype (Cdx2-FokI-BsmI) were associated with poor prognoses in colorectal cancer and lung cancer, respectively The haplotype A-T-G , sun exposure, or circulating vitamin D levels of patients. Moreover, we did not have clinical information on alcohol use, human papilloma virus, or Epstein-Barr virus infection. There are other kinds of VDR polymorphisms related to disease risks that have been reported in previous articles, and we need to expand our range of analysis in the future.There are several limitations to our study. Overall, the sample size is not large enough to detect significant differences of minor VDR polymorphisms or synergistic effects of the gene-environment and stratification by tumor stage or primary site. Although FokI T/T genotype as well as A-T-G (Cdx2-FokI-ApaI) haplotype, although the sample size is not large enough to detect significant differences of minor VDR polymorphisms or synergistic effects of the gene-environment and stratification by tumor stage or primary site.In conclusion, there were significant associations between shorter progression-free survival time in patients with HNSCC and Fok


Pseudozyma aphidis is an efficient producer of mannosylerythritol lipids exceeding concentrations of >100 g/liter from renewable feed stocks. Additionally, a biosurfactant cellobiose lipid is also secreted during nitrogen limitation. Here, we describe the sequencing of P. aphidis to unravel the genomic basis of biosurfactant metabolism in P. aphidis. Pseudozyma and Ustilago, of which Pseudozyma aphidis facilitates product concentrations of up to 165 g/liter. This species secretes a mixture of the MEL-A, -B, -C, and –D, which share a common sugar group, two fatty acid residues of medium chain length, and different numbers of acetyl groups. In Ustilago maydis, the gene cluster for MEL biosynthesis encodes the glycosyltransferase Emt1p, two acyltransferases Mac1p and Mac2p, as well as a transporter protein Mmf1p and the acetyltransferase Mat1p  belong to the most promising microbial biosurfactants and are secreted by fungi of the genera P. aphidis DSM70725. For this purpose, we sequenced the corresponding genomic DNA to approximately 90-fold coverage using the Illumina platform (HiSeq 2000), comprising a total amount of 35,141,960 reads, each 50 nucleotides in length. In addition, we also sequenced a paired-end cDNA library of the P. aphidis transcriptome comprising 48,195,420 read pairs with 2 × 95 nucleotides in length and approximately 300 nucleotides insert size (HiSeq 2000). Running the Velvet short-read assembler , revealing a G+C content of 61.2%. In a next step, we blasted (BLASTn) these nucleotide contigs against the genome of the closely related species P. antarctica T34. The total alignment length of the top BLAST hits was 14.1 Mb for 1,950 contigs, with an average identity of 97.63%. This alignment permitted an assignment to 24 supercontigs, whereas 18 remaining contigs could not be aligned. For the annotation of newly in silico-predicted genes, we applied Augustus . Searching this open reading frame (ORF) collection for homologs in the nr database of NCBI revealed 5,589 hits, with an average identity of 74.51% of the top BLAST hits . Strikingly, we identified the complete MEL biosynthesis gene cluster at the beginning of supercontig 20, which included EMT1, MAC1, MAC2, MMF1, and MAT1. All five relevant MEL genes are significantly conserved between P. antarctica T34 and P. aphidis, with similarities of 89.4%, 86.8%, 91.2%, 87%, and 86.8% at the nucleotide level, respectively. These results indicate that a similar MEL pathway exists in P. aphidis as was already shown for P. antarctica and U. maydis (Here, we describe the draft genome sequence of the MEL-producing species AWNI00000000. The version described in this paper is version AWNI01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Ghrelin, an orexigenic peptide, regulates energy balance specifically via hypothalamic circuits. Growing evidence suggest that ghrelin increases the incentive value of motivated behaviours via activation of the cholinergic-dopaminergic reward link. It encompasses the cholinergic afferent projection from the laterodorsal tegmental area (LDTg) to the dopaminergic cells of the ventral tegmental area (VTA) and the mesolimbic dopamine system projecting from the VTA to nucleus accumbens (N.Acc.). Ghrelin receptors (GHS-R1A) are expressed in these reward nodes and ghrelin administration into the LDTg increases accumbal dopamine, an effect involving nicotinic acetylcholine receptors in the VTA. The present series of experiments were undertaken directly to test this hypothesis. Here we show that ghrelin, administered peripherally or locally into the LDTg concomitantly increases ventral tegmental acetylcholine as well as accumbal dopamine release. A GHS-R1A antagonist blocks this synchronous neurotransmitter release induced by peripheral ghrelin. In addition, local perfusion of the unselective nicotinic antagonist mecamylamine into the VTA blocks the ability of ghrelin (administered into the LDTg) to increase N.Acc.-dopamine, but not VTA-acetylcholine. Collectively our data indicate that ghrelin activates the LDTg causing a release of acetylcholine in the VTA, which in turn activates local nicotinic acetylcholine receptors causing a release of accumbal dopamine. Given that a dysfunction in the cholinergic-dopaminergic reward system is involved in addictive behaviours, including compulsive overeating and alcohol use disorder, and that hyperghrelinemia is associated with such addictive behaviours, ghrelin-responsive circuits may serve as a novel pharmacological target for treatment of alcohol use disorder as well as binge eating. The cholinergic-dopaminergic reward link is an important part of the reward systems in vivo microdialysis. In addition, the role of nicotinic acetylcholine receptors, in the VTA, for this activation was investigated.Growing evidence suggest that ghrelin, a 28 amino acid gut-brain signal, which regulates energy balance specifically via hypothalamic circuits ad libitum, except during the microdialysis experiment. The Ethics Committee for Animal Experiments in Gothenburg, Sweden, has approved the experiments (permit number: 81–07 and 26–12) and all efforts were made to minimize suffering.Adult post-pubertal age-matched male Wistar rats  were used as a similar study has been reported using this strain Acylated rat ghrelin  was diluted in 0.9% sodium chloride  for peripheral  (5 ml/kg body weight) administration. The higher dose, 0.33 mg/kg, was selected since it previously has been shown to increase locomotor activity and accumbal dopamine release, induce a conditioned place preference in mice For measurements of extracellular levels (reflecting the release of the neurotransmitter) of dopamine in the N.Acc. and acetylcholine in the VTA, rats were implanted unilaterally with microdialysis probes positioned in the N.Acc. as well as in the VTA. Only rats with correct probe positions in the VTA as well as N.Acc. were included in the statistical analysis. The surgery was preformed as described thoroughly elsewhere The microdialysis technique enables measurements of neurotransmitters in awake, freely moving animals. On the day of the experiment the probe was connected to a microperfusion pump  and perfused with Ringer solution at a rate of 1.6 µl/minute. After one hour of habituation to the microdialysis set-up, perfusion samples were collected every 20 minutes. Neostigmine bromid , an acetylcholine esterase inhibitor, was added to the Ringer perfusion in the VTA throughout the entire experiment at a concentration of 0.5 µM to improve the detection of acetylcholine. It has previously been shown that this dose does not affect the extracellular concentrations of dopamine in the N.Acc. The first series of experiments were undertaken to investigate if peripheral ghrelin activates the cholinergic-dopaminergic reward link in the brain. The effects of two different doses of ghrelin (0.167 or 0.33 mg/kg i.p.) compared to vehicle (sodium chloride) on ventral tegmental acetylcholine and accumbal dopamine in rats were investigated. The doses used are from a narrow dose interval and the experiment should therefore not be considered as a dose response study. The lower dose (0.167 mg/kg) has previously been shown to release dopamine in the shell of N.Acc. in rats i.e. vehicle-vehicle; vehicle-ghrelin; JMV2959-vehicle; JMV2959-ghrelin. The following eight samples were collected and analysed.The second series of experiments were conducted to confirm that the ability of ghrelin to activate the cholinergic-dopaminergic reward link is mediated via GHS-R1A. The higher dose of ghrelin (0.33 mg/kg i.p.) was used since this dose is commonly used in rats and produces a robust activation of the cholinergic-dopaminergic reward link. In these experiments, conducted in separate rats, we examine the effect that the GHS-R1A antagonist JMV2959 has on ghrelin-induced release of acetylcholine and dopamine in the VTA and N.Acc., respectively. The baseline acetylcholine or dopamine levels were defined as the average of three consecutive samples before the first drug challenge, and the increase in acetylcholine or dopamine was calculated as the percentage increase from baseline. After the baseline samples, rats were injected with JMV2959  or vehicle. 20 minutes later this was followed by a ghrelin  or vehicle injection, creating four treatment groups, i.e. vehicle-vehicle; vehicle-ghrelin; mecamylamine-vehicle; mecamylamine-ghrelin. The increase in acetylcholine or dopamine was calculated as the percentage increase from baseline. The following eight samples were collected and analysed.In subsequent experiments we investigated if ghrelin locally into the LDTg activates the cholinergic-dopaminergic reward link and if this activation is mediated via local nicotinic acetylcholine receptors in the VTA. Therefore, we examined whether ghrelin administered locally into the LDTg produces a concomitant release of acetylcholine in the VTA and dopamine in the N.Acc. in rats. In addition, we investigated the effect of local perfusion of the unselective nicotinic acetylcholine receptor antagonist, mecamylamine, into the VTA on ghrelin (administered into the LDTg) induced synchronous neurotransmitter release. The baseline acetylcholine or dopamine levels were defined as the average of three consecutive samples before the first drug challenge. Mecamylamine (300 µM) or vehicle (Ringer) was perfused locally into the VTA via the probe for 40 minutes and thereafter ghrelin (1 µg in 0.5 µl) or vehicle (Ringer) was administered locally into the LDTg, creating four treatment groups, After the microdialysis experiments were completed, the locations of the probes and guide cannula were verified. The rats were decapitated, probes were perfused with pontamine sky blue 6BX to facilitate probe localization, and the brains were mounted on a vibroslice device . The brains were cut in 50 µm sections and the location of the probes and guide cannula were determined by gross observation using light microscopy. The exact positions were verified The acetylcholine levels in the dialysates were determined by means of liquid chromatography/tandem mass spectrometry (LC-MS/MS), essentially as described elsewhere The dopamine levels in the dialysates were determined by means of HPLC with electrochemical detection (HPLC-EC). The analysis was preformed as described thoroughly elsewhere All microdialysis experiments were evaluated by a two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test for comparisons between different treatments and specifically at given time points. Data are presented as mean ± SEM.Here we initially showed that ghrelin (0.167 and 0.33 mg/kg) increases ventral tegmental and accumbal dopamine concomitantly in rats.P = 0.0105 and P = 0.0015, respectively)  = 8.42, P = 0.0032; time F = 2.54, P = 0.0040; treatment x time interaction F = 1.68, P = 0.0305). This increase was significant at time interval 20 minutes (P<0.01) and 100 minutes (P<0.05) (for ghrelin 0.33 mg/kg versus vehicle). No statistically significant difference was observed between the two different doses of ghrelin (0.33 mg/kg and 0.167 mg/kg) (P = 0.3212) (data not shown).Both doses of ghrelin (0.167 and 0.33 mg/kg) increased ventral tegmental acetylcholine release relative to vehicle treatment (P = 0.0062 and P = 0.0003 respectively)  = 11.56, P = 0.0008; time F = 3.26, P = 0.0003; treatment x time interaction F = 2.64, P = 0.0001). This increase was significant at time intervals 60–80 minutes (P<0.001), 140 minutes (P<0.01) and 180 minutes (P<0.05) (for ghrelin 0.33 mg/kg versus vehicle). No statistically significant difference was observed between the two different doses of ghrelin (0.33 mg/kg and 0.167 mg/kg) (P = 0.1473) (data not shown).Both doses of ghrelin (0.167 and 0.33 mg/kg) increased accumbal dopamine release relative to vehicle treatment .The second series of experiments first verified our initial findings that ghrelin concomitantly increases ventral tegmental acetylcholine and accumbal dopamine, and secondly showed that a GHS-R1A antagonist blocked this dual neurotransmitter release.P = 0.0023) and that this increase was attenuated by pre-treatment with JMV2959 (3 mg/kg) (P = 0.0003)  = 8.63, P = 0.0006; time F = 6.43, P<0.0001; treatment x time interaction F = 3.30, P<0.0001). This attenuation was significant at time intervals 40 minutes (P<0.001), 60 and 140 minutes (P<0.001), 160–180 minutes (P<0.05) (for vehicle-ghrelin versus JMV2959-ghrelin). No difference was observed between vehicle-vehicle and JMV2959-ghrelin treatment (P = 0.3365). JMV2959 had no effect on acetylcholine release per se (P = 0.1116)  increased ventral tegmental acetylcholine release relative to vehicle treatment ( 0.1116) .Given that acetylcholine and dopamine was measured in one and the same rats the number of rats induced in the statistical analysis was identical .P<0.0011) and this increase was attenuated by pre-treatment with JMV2959 (3 mg/kg) (P = 0.0120)  = 5.10, P = 0.0079; time F = 2.25, P = 0.0101; treatment x time interaction F = 2.57, P<0.0001). This attenuation was significant at time interval 20 minutes (P<0.001), 40–60 minutes (P<0.01), and 100 minutes (P<0.05) (for vehicle-ghrelin versus JMV2959-ghrelin). No difference was observed between vehicle-vehicle and JMV2959-ghrelin treatment (P = 0.3205). JMV2959 had no effect on dopamine release per se (P = 0.6284) (Ghrelin (0.33 mg/kg) increased accumbal dopamine release relative to vehicle treatment ( 0.6284) .Here we initially showed that ghrelin locally into the LDTg increases ventral tegmental and accumbal dopamine concomitantly in rats. In addition we showed that the unselective nicotinic acetylcholine receptor antagonist mecamylamine locally perfused into the VTA blocks the ability of ghrelin (administered into the LDTg) to increase accumbal dopamine, but not ghrelin induced acetylcholine release in the VTA .P = 0.0435, vehicle-vehicle vs. vehicle-ghrelin) = 3.85, P = 0.0166; time F = 24.29, P<0.0001; treatment x time interaction F = 4.83, P<0.0001). The ability of ghrelin administered into the LDTg to increase ventral tegmental acetylcholine release was significant at time intervals 20–40 minutes (P<0.001) and at 60 minutes (P<0.01) (vehicle-vehicle versus vehicle-ghrelin). Pre-treatment of mecamylamine into the VTA did not affect the ability of ghrelin into the LDTg to increase ventral tegmental acetylcholine release  and this was significant at time intervals 20–40 minutes (P<0.001) and at 60 minutes (P<0.01) (vehicle-vehicle versus mecamylamine-ghrelin). Mecamylamine had no effect on acetylcholine release per se (P = 0.6580)  increased ventral tegmental acetylcholine release relative to vehicle treatment ( 0.6580) .P = 0.0207, vehicle-vehicle vs. vehicle-ghrelin) = 5.11, P = 0.0044; time F = 6.31, P<0.0001; treatment x time interaction F = 2.52, P<0.0001). This was significant at time interval 20–60 minutes (P<0.01) (vehicle-vehicle versus vehicle-ghrelin). This increase was attenuated by pre-treatment with mecamylamine (300 µM) . This attenuation was significant at time intervals 20 minute (P<0.01), 40 minute (P<0.001) and 60 minute intervals (P<0.05) (vehicle-ghrelin versus mecamylamine-ghrelin). Mecamylamine had no effect on dopamine release per se (P = 0.1803)  increased accumbal dopamine release relative to vehicle treatment ( 0.1803) .Given that acetylcholine and dopamine was measured in one and the same rats the number of rats induced in the statistical analysis was identical .In the present series of experiments we show for the first time that systemic ghrelin administration increases ventral tegmental acetylcholine as well as accumbal dopamine release concomitantly in rats. Peripheral administration of the GHS-R1A antagonist, JMV2959, blocks this synchronous neurotransmitter release, suggesting that ghrelin reaches the brain and activates the cholinergic-dopaminergic reward link via GHS-R1A. In addition, local ghrelin administration into the LDTg increases VTA-acetylcholine and N.Acc.-dopamine; perfusion of the unselective nicotine acetylcholine receptor antagonist (mecamylamine) into the VTA blocks the ghrelin-induced release of accumbal dopamine, but not that of acetylcholine. Collectively, these data indicate that ghrelin activates the LDTg causing a release of acetylcholine in the VTA, which via activation of local nicotinic acetylcholine receptors, causes a release of accumbal dopamine.Growing evidence has implied that the cholinergic-dopaminergic reward link mediates the reinforcing properties of natural as well as artificial rewards. Specifically, infusion of a nicotinic agonist into the VTA increased the extracellular dopamine levels in the N.Acc. The GHS-R1A antagonist, JMV2959, blocks the ghrelin-induced dual increase of acetylcholine in the VTA and dopamine in the N.Acc., implying that peripheral ghrelin reaches reward nodes and activates the cholinergic-dopaminergic reward link via local GHS-R1A. In support of this, ghrelin passes the blood brain barrier The present study also show that the ability of ghrelin, administered into the LDTg, to increase accumbal dopamine, but not VTA-acetylcholine, is blocked by perfusion of the unselective nicotinic acetylcholine receptor antagonist, mecamylamine, into the VTA. Previously, alpha3beta2, beta3 and alpha6 nicotinic acetylcholine receptors subtypes in the VTA appear to be critical for this activation In summary, the present study provides proof of our concept that ghrelin activates the cholinergic-dopaminergic reward link, which is intimately involved in natural- as well as drug-induced reward and also associated with addictive behaviours. These data may have clinical implications since hyperghrelinemia is associated with addictive behaviours including binge eating and alcohol use disorder. Thus, anorectic and bulimic patients of the binge eating type have higher levels of ghrelin in the plasma than their non-binge eating counterparts, and the frequencies of binge eating correlate positively with plasma ghrelin levels


Synapsins are synaptic vesicle (SV) proteins organizing a component of the reserve pool of vesicles at most central nervous system synapses. Alternative splicing of the three mammalian genes results in multiple isoforms that may differentially contribute to the organization and maintenance of the SV pools. To address this, we first characterized the expression pattern of synapsin isoforms in the rat calyx of Held. At postnatal day 16, synapsins Ia, Ib, IIb and IIIa were present, while IIa—known to sustain repetitive transmission in glutamatergic terminals—was not detectable. To test if the synapsin I isoforms could mediate IIa-like effect, and if this depends on the presence of the E-domain, we overexpressed either synapsin Ia or synapsin Ib in the rat calyx of Held via recombinant adeno-associated virus-mediated gene transfer. Although the size and overall structure of the perturbed calyces remained unchanged, short-term depression and recovery from depression were accelerated upon overexpression of synapsin I isoforms. Using electron microscopic three-dimensional reconstructions we found a redistribution of SV clusters proximal to the active zones (AZ) alongside with a decrease of both AZ area and SV volume. The number of SVs at individual AZs was strongly reduced. Hence, our data indicate that the amount of synapsin Ia expressed in the calyx regulates the rate and extent of short-term synaptic plasticity by affecting vesicle recruitment to the AZ. Finally, our study reveals a novel contribution of synapsin Ia to define the surface area of AZs. Synapsins are a family of abundant neuron-specific phosphoproteins whose expression varies across synapse types and brain regions with synapsin Ia, Ib, IIa, IIb, and IIIa being most abundant . This synapse provides a well-suited model system to investigate SV protein organization and function in close proximity to the release sites. The calyx of Held harbors more than 600 active zones (AZ)  were used for expressing synapsin isoforms in the medial nucleus of the trapezoid body (MNTB). Plasmids expressing synapsin isoforms, tagged at the N-terminus with enhanced green/yellow fluorescent proteins (EGFP/EYFP), were constructed as follows: EGFP, EYFP-synapsin Ia, or synapsin Ib was excised from pEGFP, EYFP-synapsin Ia, or synapsin Ib-C1, respectively, .Experiments were conducted in accordance with the German animal welfare guidelines (TierSchG) and were approved by our local animal care and use committee (Regierungspräsidium Karlsruhe). Sprague-Dawley rats from either sex were used for all experiments. Seven rats were injected with mGFP for detection of endogenous synapsin isoforms. Four rats were co-injected with mGFP and EYFP-synapsin Ia, five rats co-injected with mGFP and EYFP-synapsin Ib, and five rats injected with mGFP were used for fluorescent structural analysis. Seven rats injected with EYFP-synapsin Ia, 14 rats injected with EYFP-synapsin Ib and 22 non-injected wild type rats (controls) were used for electrophysiology. Two rats injected with EGFP-synapsin Ia and two rats injected with EGFP were used for electron microscopy (EM) analysis. With regard to immunohistochemical and morphological analyses, we define rats expressing mGFP/EGFP as control rats (CTRL) to which we compare rats overexpressing EYFP-synapsin Ia, or synapsin Ib. P16–18 animals were used to circumvent the strong myelination present in the brain stem of adult rats, which makes x, y positions injections were made at depth (z) of 0.45 mm ventral from the bregma and axially (Ax)at −8.0 to −9.5 mm.rAAV1/2 particles were delivered to the anterior ventral cochlear nucleus (aVCN) of rats at postnatal day 6 via stereotaxic injection as reported previously , followed by 40 ml 4% paraformaldehyde in PBS. The brains were extracted and post-fixed in 4% paraformaldehyde overnight at 4°C. The rat brain stem was embedded in 3% agarose and 50 μm-thick coronal sections were cut on a vibratome . Prior to staining, sections were washed 3 × 10 min in PBS and incubated 60 min at room temperature  in PBS containing 5% normal goat serum and 1% TritonX-100. The primary antibodies were diluted in 4°C PBS with 1% normal goat serum and 0.5% TritonX-100, and incubated overnight at RT. The secondary antibodies, conjugated to Alexa fluorophores , were diluted in the same buffer as primaries, and incubated for 90 min at RT in the dark. After 3 × 10 min washing in 4°C PBS, the sections were rinsed with distilled H2O and embedded in SlowFade® Gold antifade reagent . In case of synapsin IIIa, normal goat serum was exchanged with fetal bovine serum .Ten days after injection, rats were anesthetized with a lethal dose of sodium-pentobarbital  and transcardially perfused with 40 ml phosphate-buffered saline  and 476 nm, 488 nm, 514 nm, 568 nm and 647 nm laser lines to excite the respective fluorophores. After collecting images with a voxel size of 91.71 nm × 91.71 nm axial and 150 nm vertical dimensions, data was deconvolved using Huygens software (Scientific Volume Imaging). In experiments involving imaging of mGFP and EYFP in the same slice preparation, spectral unmixing of the acquired data was performed via the Channel Dye Separation Application of the Leica Application Software, prior to deconvolution. For the purpose of defining reference regions we imaged terminals expressing either mGFP or EYFP-synapsin Ia/Ib. Calyces in the ipsilateral (non-infected) MNTB were used as a reference for vGluT1 staining. Reference and experimental images were acquired with the same microscope settings.Excision of calyx-specific immunosignals and 3D reconstruction of image data was performed with the visualization software AMIRA 4.1.1 (Visage Imaging) as described previously , the pH was 7.3 and density was 315–320 mOsm. Slices were incubated in normal artificial cerebrospinal fluid (aCSF) for ~ 60 min at 35°C, then used thereafter at RT. Normal aCSF contained (in mM) 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, and 1 MgCl2, pH 7.3 after bubbling with carbogen, density 315–320 mOsm. Strychnine (0.5 μM), SR9551 (10 μM), and APV (50 μM) were also added to the recording bath solution, which perfused the slice at 1-2 mL/min. All materials were purchased from Sigma Aldrich, unless stated otherwise.Acute transverse 200 μm-thick slices were made from P16–18 rat brainstem, containing intact ventral stria and MNTB. Slices were made on a vibratome , at 4°C. Slicing media contained (in mM): 85 NaCl, 2.5 KCl, 25 glucose, 25 NaHCOMNTB cells were visualized at 60x, using an EM CCD camera , and gradient contrast optics on a Zeiss microscope . Infected calyx terminals were identified by strong fluorescence signal opposing intact principal cell somata. MNTB cells were additionally preselected based on the presence of extracellular presynaptic and postsynaptic waveforms during afferent stimulation, showing intact axonal connectivity  amplitude exceeded 18 nA, in which case holding potential was reduced to -30 mV to reduce the driving force. Because the recordings of evoked EPSCs (eEPSC) were made at multiple holding potentials, direct comparison is unwarranted without biased selection. To assist in cross-cell normalization, EPSCs were evoked in multiple cells at both Vhold −70 and −30 mV holding potential (data not shown). From the resulting IV relationship a scaling factor of 0.025 ± 0.001 nA /mV for the eEPSCs amplitude was calculated, and applied to generate a predicted eEPSC magnitude at Vhold -70 mV. Access resistance was in all cases ≤6 MΩ, and compensated 85–94%, such that residual series resistance (Rs) <0.5 MΩ. Leak current was usually less than 100 pA, and in no cases exceeded 200 pA.Whole-cell patch clamp recordings from the MNTB principal cell were made using 1.5 mm outer diameter filamented borosilicate glass , pulled to tip resistances of 1.8–2.5 MΩ on a Sutter P-97 puller . Pipette internal solution consisted of (in mM): 130 Cs-gluconate, 10 CsCl, 5 NaEPSCs were evoked via midline stimulation using parallel Pt-Ir bipolar electrodes . Stimuli were applied with an optically isolated bipolar stimulator , using 100 μs duration pulses. Stimulation amplitude was set to 1 V above threshold (1.0–3.5 V). Stimulus trains were generally repeated 3–5 times per cell, with a 30 s interval between trains, and the mean initial EPSC magnitude was normalized. The paired-pulse ratio (PPR), rate of depression, and steady-state level of the depressed synapse were then measured per cell, from the averaged traces  was estimated according to . Data were filtered on-line at 2.9 kHz. Data were corrected off-line for voltage errors due to uncompensated series resistance  and 40 μl 1 M NaOH (pH 7.6). A Leica DM IRB microscope equipped with a 40x oil immersion objective (NA: 0.75–1.25) was used for photooxidation. The light path aperture was closed to minimum and had a diameter of 150 μm. The region of interest was illuminated with wavelength 488 nm for 5 min with NA 0.75, followed by 5 min with NA 1.0 and then by 5 min with NA 1.25 and fine precipitates were observed in the exposed area. The tissue was then washed 2× in 50 mM Tris-HCl and incubated in 0.1 M cacodylic acid buffer buffer pH 7.4. The MNTB area containing the photooxidized terminals was dissected out and post-fixed in a mixture of 2% potassium potassium ferrocyanide and 2% osmium tetroxide for 120 min. After 3 washing steps in distilled water they were dehydrated in an ascending alcohol series and stored overnight in a 1:1 mixture of propylene oxide and epoxy . The tissue was then transferred into fresh resin and polymerized at 60°C for 36 h. After polymerization we cut 20–30 serial sections per sample with a thickness of ~ 35 nm on an ultramicrotome. The resulting ribbons were attached to a silicon wafer , counterstained with uranyl acetate and lead citrate, and prepared for imaging following published procedures  analysis of the data was made by alignment of the serial sections using OpenCAR software, version 1.5.79  with significance level p < 0.05, unless otherwise indicated. Mean ±s.e.m. values are shown in summary data plots. Photomicrographs were generated with Image J 1.41o, AMIRA 4.1.1 and Adobe Photoshop CS4, version 10.0. Final images were aligned and labeled in Adobe Illustrator CS4, version 13.0.0.Statistical analysis and curve fitting was performed using Prism 5.0b . Curve fits used built-in functions, were performed on group mean values unless otherwise indicated, with outlier exclusion and with minimal parameter constraints. Variability arises mostly between individual calyces and to a lesser degree from individual animals . Staining with an antibody recognizing the E-domain specifically visualizes the a-isoforms . In summary, synapsin Ia, Ib, IIb and IIIa are endogenously expressed in the rat calyx of Held at P16 and are co-expressed within the same volume of the presynaptic terminal, apart from a possible subcompartmentalization of synapsin Ia and Ib expression. In contrast, the synapsin IIa isoform is not present in appreciable quantity in the calyx of Held.Next, we tested if all synapsin isoforms occupy the same presynaptic volume. For this purpose we examined the localization of specific isoforms with respect to the distribution of synapsin I Figure . Synapsi Figures . Becauset-test P = 0.02, 0.03, 0.01, respectively). Although the calyx of Held expresses both vGluT1 and vGluT2, their expression patterns are largely overlapping at the age examined here . Quantification of the vGluT1 immuno-signal did not reveal an effect of synapsin Ia or synapsin Ib overexpression on the level of vGluT1  were recorded in MNTB principal cells for 2–4 min after establishing the whole-cell configuration, and prior to any high-frequency stimulation. The properties of spEPSC  were not changed by the overexpression of synapsin Ia or synapsin Ib Table . Hence, Evoked EPSCs (eEPSCs) were measured in response to afferent fiber stimulation at 0.1 Hz, evoking an all-or-none response in the MNTB principal cells. Basal eEPSC magnitude  did not significantly differ between control and synapsin overexpression conditions . Synapsin Ib overexpression likewise sped depression, with a time constant of 32.6 ± 2.1 ms , and a steady state of 18.2 ± 1.4% . Thus, overexpression of either synapsin Ia or synapsin Ib resulted in faster and more severe depression at 100 Hz compared to control terminals.Stimulation at higher frequency (100 Hz) resulted in strong depression, which was fit by a monoexponential function for responses in individual cells from the 2–50th stimuli Figure . The firfast) of short-term depression in control synapses were τfast = 7.4 ± 1.0 ms (70.0 ± 1.3%), and τslow = 33.5 ± 2.8 ms. Overexpression synapsin Ia resulted in significantly faster τfast  and τslow  but a similar relative weight (68.0 ± 2.3%). The mean depression time constant (τmean) was calculated according to the formula: [τmean = τ*fast(%τfast) + τ*slow(100%–%τfast)]. τmean for control synapses was 15.2 ± 1.2 ms. Synapsin Ia overexpression resulted in a significantly faster mean depression time constant . Notably, overexpression of synapsin Ib did not lead to acceleration of depression—τfast was 6.01 ± 0.66 ms with relative weight 73.3 ± 1.7%, and τslow of 31.1 ± 3.0 ms. This resulted in τmean of 12.7 ± 1.0 ms  and to 5.6 ± 0.8% by synapsin Ib overexpression . There was, however, no effect of synapsin Ib overexpression on the PPR at any ISI.Using the data presented above, we examined the PPR for potential differences in facilitation or depression early in the stimulus train, due to synapsin I overexpression Figures . ConsistPr of synapses overexpressing synapsin Ia, but not synapsin Ib, relative to controls /(EPSC1-EPSCss), where EPSCn represents the measured EPSC after the depleting train, EPSCss is the steady-state EPSC integral, to account for different levels of depression between the control and infected terminals, and EPSC1 is the first response in the depletion train.Following short trains at 100 Hz, recovery of the partially-depleted pool of release-competent vesicles was assayed, using single test pulses at variable intervals following the end of the stimulus train . Overexpression of either synapsin Ia or Ib did not significantly affect the time course of recovery in this protocol, with synapses overexpressing synapsin Ia recovering at a rate K = 0.23 ± 0.01 s−1, and synapsin Ib-overexpressing synapses recovering at K = 0.22 ± 0.02 s−1.For a short train (6 pulses at 100 Hz), transmission was depressed to ~ 50% in control synapses, and recovered in all conditions with a monoexponential time course (K = 0.25 ± 0.03 s−1 (r2 > 0.995); however, overexpression of each synapsin I isoform resulted in significantly faster recovery to the initial EPSC magnitude. It is evident that synapsin Ia overexpression significantly speeds the recovery of EPSCs . Synapsin Ia overexpression sped recovery due to an increase in the weight of the fast component (Kfast = 6.83 ± 1.58 s−1), to 37.7 ± 2.7% of recovery , and a (Kslow = 0.36 ± 0.04 s−1). Recovery in synapsin Ib-expressing terminals resulted in Kfast = 7.19 ± 2.73 s−1  and Kslow = 0.20 ± 0.03 s−1, which was significantly slower than the synapsin Ia slow component . By contrast, control data could not be fit double-exponentially without constraints. To be able to make a direct comparison with the control the data from the infected terminals was fit monoexponentially with rate constants of K = 0.25 ± 0.03 s−1 for control terminals and K = 0.59 ± 0.1 s−1 for synapsin Ia and K = 0.31 ± 0.06 s−1 for synapsin Ib overexpressing calyces , we counted 1.7 ± 0.3 docked SVs per AZ in control terminals and only 0.6 ± 0.2 docked SVs per AZ in synapsin Ia-overexpressing calyces . SVs in synapsin Ia-overexpressing terminals were also individually smaller, having an average diameter of 37.57 ± 0.33 nm, while the SVs in control terminals had diameters of 40.2 ± 0.38 nm .We next examined the SV distribution in close proximity to the release sites. We traced 33 AZs in EGFP-overexpressing calyces and 32 AZs in EGFP-synapsin Ia-overexpressing terminals. AZs of both EGFP and EGFP-synapsin Ia-overexpressing calyces had associated SV clusters Figures . We perf2 vs. 0.124 ± 0.012 μm2 , while AZ size did not influence the number of SVs for synapsin Ia overexpression  GFP-synapsin is properly targeted to the presynaptic terminals as shown in Figure Aplysia sensorimotor neuron co-cultures has been reported to enlarge the reserve pool of SVs (Fioravante et al., Our findings strongly suggest that synapsin I isoforms mediate SV distribution within the presynaptic terminal. Both on the scale of entire presynaptic terminals, and on the scale of clusters associated with single AZs, overexpression of synapsin Ia causes vesicle redistribution Figures , 9. OverOverexpression of synapsin Ia did not cause noticeable alterations in the overall ultrastructure of the calyceal segments examined with EM Figure . HoweverOverexpression of synapsin Ia led to alterations in the size of SVs and AZ—SVs were smaller and AZ surface area was halved Figure . InteresOverexpression of synapsin I isoforms caused a significant acceleration of the decay times and produced deeper steady-state depression, consistent with previous work (Gitler et al., 2+—calmodulin—Munc13-1 mechanism (Lipstein et al., 2+-modulated SV endocytosis or recycling (Bloom et al., 2+ signal into faster SV retrieval and recycling.Recovery from short-term depression was accelerated in calyces overexpressing synapsin Ia, but not synapsin Ib. This appears inconsistent with the accelerated short-term depression observed most strongly after synapsin Ia overexpression Figure , yet theStudy concept and design: Mariya Vasileva, Robert Renden, Thomas Kuner, Daniel Gitler. Acquisition of data: Mariya Vasileva, Robert Renden, Heinz Horstmann. Analysis and interpretation of data: Mariya Vasileva, Robert Renden, Thomas Kuner. Drafting of the manuscript: Mariya Vasileva, Robert Renden, Thomas Kuner. Critical revision of the manuscript for important intellectual content: Thomas Kuner, Daniel Gitler. Obtained funding: Thomas Kuner, Daniel Gitler, Robert Renden. Study supervision: Thomas Kuner.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.


Immunoglobulin E (IgE) binds to high affinity receptor FcεRI numerously expressed on mast cells. Recent findings have revealed that IgE by itself may regulate various aspects of mast cell biology, however, detailed data is still limited.in vivo differentiated mature tissue mast cells isolated from rat peritoneal cavity. Mast cells were exposed to IgE alone and then the release of preformed and de novo-synthesized mediators, surface FcεRI expression and mast cell migratory response were assessed. IgE by itself was found to up-regulate FcεRI expression and activate mast cells to degranulation, as well as de novo synthesis and release of cysteinyl leukotrienes and TNF. We have provided evidence that IgE alone also amplified spontaneous and CCL5- or TNF-induced migration of mast cells. Importantly, IgE was effective only at concentrations ≥ 3 µg/mL. A molecular basis investigation using an array of specific inhibitors showed that Src kinases, PLC/PLA2, MAP kinases (ERK and p38) and PI3K were entirely or partially involved in IgE-induced mast cell response. Furthermore, IgE alone stimulated the phosphorylation of MAP kinases and PI3K in rat mast cells.Here, we have examined the influence of IgE alone, used at different concentrations, on mast cell activity and releasability. For the study we have employed in vivo IgE is one of the important factors modulating mast cell biology within tissues.Our results clearly demonstrated that IgE by itself, at higher concentrations, influences mast cell activity and releasability. As there are different conditions when the IgE level is raised it might be supposed that  Under physiological conditions, immunoglobulin E (IgE) synthesis and, hence its concentration in the blood or within tissues is constantly low via their involvement in angiogenesis, tissue remodeling and repair, but also are key players in inflammatory processes and the host response to pathogens. Moreover, mast cells play an important role in the course of many diseases, other than IgE-dependent hypersensitivity reactions, for example, atherosclerosis, rheumatoid arthritis, congestive heart failure, malignancies, Crohn's disease and pulmonary fibrosis 2+. Of note, the majority of studies were carried out using cell lines differentiated in vitro , which differ with respect to phenotype and activity from mast cells, which develop and mature under influence of microenvironmental factors. It should be also emphasized that the available data concerning the influence of IgE exposure on mast cell response and activity still remains limited.IgE binds with high affinity to the FcεRI abundantly expressed on mast cells. Mast cells are widely distributed throughout the body and are the source of large numbers of biologically active mediators. Mast cell-derived mediators exert diverse proinflammatory, anti-inflammatory, and/or immunoregulatory effects and modulate the activity of many cell populations. Thus, mast cells participate not only in maintaining homeostasis mainly de novo synthesis. Another important question to clarify was whether IgE alone affects spontaneous migratory response of mast cells, as well as migration induced by strong mast cell chemotactic factors such as CCL5 and TNF via FcεRI, i.e. mitogen-activated protein (MAP) kinases  and p38 kinase), Src kinases, phosphatidylinositol 3-kinase (PI3K) and phospholipase (PL)C/PLA2.In the present study we scheduled to examine the direct impact of IgE alone, used at different concentrations, on various effector functions of fully mature rat peritoneal connective tissue mast cells. To this end, we examined the IgE-induced mast cell preformed mediators release, arachidonic acid metabolite generation and tumor necrosis factor (TNF) 2, CaCl2, 2-hydroxyethylpiperazine-N'-ethanesulphonic acid (HEPES), NaOH, glucose, HCl, o-phthaldehyde (OPT), Percoll, hematoxylin, toluidine blue, trypan blue, bovine serum albumin (BSA), PLC/PLA2 inhibitor (U73122), PI3K inhibitor (LY294002), Src kinase inhibitor , RIPA buffer and laminin from human placenta were obtained from Sigma-Aldrich . ERK inhibitor (PD98059) and p38 kinase inhibitor (SB203580) were purchased from InvivoGen . Recombinant rat (rr) tumor necrosis factor (TNF) was obtained from R&D Systems , rrCCL5, and mouse anti-rat IgE monoclonal IgG1 antibodies (anti-IgE) were purchased from AbD Serotec . Mouse monoclonal IgG1 anti-FcεRI and anti-PI3K antibodies were obtained from Abcam Inc.  and purified rat myeloma IgE was obtained from Invitrogen, Life Technologies . Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse polyclonal antibodies, purified mouse monoclonal IgG1 antibodies (isotype control), and BD CellFIX were purchased from BD Biosciences . The 48-well Boyden microchamber as well as the 8-μm-pore-size polycarbonate filters were purchased from Neuro Probe . Rat TNF and cysLT specific immunoassay kits were purchased from Gen-Probe Inc.  and Cayman Chemical , respectively. The RNeasy Mini Kit was obtained from Qiagen . The High Capacity cDNA Reverse Transcription Kit, TaqMan® probes dyed 6-carboxyfluorescein (6-FAM) (rat TNF Rn01525859 and rat Actb Rn00667869) and TaqMan® Gene Expression Master Mix were purchased from Applied Biosystems  and bicinchoninic acid (BCA) Protein Assay Kit was obtained from Pierce . The Western Lightning luminol-based enhanced chemiluminescence (ECL) Pro system was purchased from Perkin-Elmer . Anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2, anti-phospho-p38 (Tyr-182) and anti-p38 as well as horseradish peroxidase (HRP)-labeled donkey anti-goat IgG, goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP all were obtained from Santa Cruz Biotechnology Inc. . Anti-phospho-PI3K (p85 Tyr458/p55 Tyr199) antibodies were obtained from Thermo Scientific .Dulbecco's Modified Eagle's Medium (DMEM), Hank's balanced salt solution (HBSS), sodium bicarbonate, fetal calf serum (FCS), gentamicin and glutamine were purchased from GIBCO, Life Technologies . NaCl, KCl, MgClad libitum. Isoflurane-induced anesthesia was carried out prior to animal decapitation. All efforts were made to minimize animal suffering. Animal experiments were approved by the Local Ethics Committee for Experiments on Animals of the Medical University of Łódź . All manipulations performed on animals were in accordance with university guidelines.Mast cells were collected from peritoneal cavities of female Wistar rats weighing ∼250 g, aged 3–4 months, bred in the animal quarters of the Medical Faculty of the Medical University of Łódź. Standard storage conditions for animals were provided, i.e. room temperature (20±2°C), artificial lighting for 12 h and 12 h of darkness, in metal cages, 5 rats in each. The animals were fed with LSM Murigran granulated fodder for rodents and water 2, 1 mM CaCl2, 10 mM HEPES, 5.6 mM glucose, and 1 mg/mL BSA (for histamine release assay and cysLT synthesis measurement), to obtain mast cell concentration of 1.5×106 cells/mL. Mast cell were prepared with purity > 98%, as determined by toluidine blue staining.Peritoneal cell suspensions were obtained from peritoneal cavities by lavage with 50 mL of 1% HBSS supplemented with 0.015% sodium bicarbonate. Peritoneal cell suspension was washed twice  in complete (c)DMEM containing DMEM supplemented with 10% FCS, 10 µg/mL gentamicin, and 2 mM glutamine. In order to collect purified mast cells, peritoneal cells were resuspended in 72.5% isotonic Percoll solution and centrifuged . After being washed twice , mast cells were counted and resuspended in an appropriate volume of cDMEM  or in medium for rat mast cells, containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgClPurified mast cells were incubated with IgE at final concentrations of 0.1, 0.5, 1, 1.5, 3, or 5 µg/mL, or without IgE (negative control) in a water bath for 1 h at 37°C with constant stirring. In every experiment a positive control for the determination of anti-IgE-induced histamine release from native mast cells  was included. For time-course experiments mast cells were incubated with IgE at final concentrations of 5 µg/mL for 0, 5, 10, 30, or 60 min. After incubation the reaction was stopped by adding 1.9 mL of cold medium for rat mast cells and the release of histamine was measured using spectrofluorometric method, as described previously Purified mast cells were incubated with IgE at final concentrations of 0.1, 0.5, 1, 1.5, 3, or 5 µg/mL, or without IgE (negative control) in a water bath for 1 h at 37°C with constant stirring. In every experiment a positive control for the determination of native mast cell cysLT synthesis in response to anti-IgE stimulation  was included. The supernatants were collected by centrifugation  and analyzed for cysLT by an ELISA commercial kit according to the manufacturer's instructions. The sensitivity of this assay was < 13 pg/mL.2. Native mast cells were treated with anti-IgE at concentration of 5 µg/mL under the same experimental conditions (positive control). The supernatants were collected by centrifugation  and analyzed for TNF by an ELISA commercial kit according to the manufacturer's instructions. The sensitivity of this assay was < 20 pg/mL.Purified mast cells were incubated with IgE at final concentrations of 0.1, 0.5, 1, 1.5, 3, or 5 µg/mL, or without IgE (negative control) for 6 h at 37°C in a humidified atmosphere with CO2. Total RNA was isolated from mast cells using an RNeasy Mini Kit. Synthesis of cDNA was performed using High Capacity cDNA Reverse Transcription Kit. For qRT-PCR, TaqMan® probes and TaqMan® Gene Expression Master Mix were used. All reactions were performed with the use of 7900 HT Fast Real-Time PCR System (Applied Biosystems). The expression of TNF mRNA was corrected by normalization based on the transcript level of the housekeeping gene rat Actb. All qRT-PCRs were conducted in triplicate, and the changes in gene expression are presented as fold-increases relative to non-stimulated mast cells.Purified peritoneal rat mast cells were stimulated with IgE or anti-IgE (positive control) both at 5 µg/mL for 0, 15, 30, 60, 180, and 360 min at 37°C in a humidified atmosphere with 5% CO2. After incubation, the non-migrating cells were removed from the upper surface of the filter by scraping, and migrating cells adherent to the lower surface of the membrane were fixed in 99.8% ethanol, washed in distilled water, stained for 10 min with hematoxylin, cleared in distilled water, and mounted on microscope slides. Mast cell migration was quantified by counting the total number of cells migrating through the filter in 10 high power fields (HPF).Mast cell migration was analyzed in a 48-well Boyden microchamber using 8-μm-pore-size polycarbonate filters. The filters were coated overnight at room temperature with laminin at a concentration of 100 µg/mL and air dried for at least 1 h before use. Purified mast cells were preincubated with IgE at 1 µg/mL and 5 µg/mL or medium alone (native mast cells) for 1 h at 37°C with constant stirring. After preincubation, mast cells were washed in cDMEM . Next, 30 µL of rrTNF at 10 ng/mL, rrCCL5 at 100 ng/mL or medium alone (spontaneous migration) was placed into the lower compartment of a microchamber, and 50 µL of native or IgE-coated mast cells was pipetted into the upper compartments. The mast cells were incubated in the chemotaxis chamber for 3 h at 37°C in a humidified incubator with 5% CO2 inhibitor (U73122) was used at a concentration of 0.1 µM, ERK kinase inhibitor (PD98059) was used at a concentration of 100 µM, PI3K inhibitor (LY294002) was used at a concentration of 50 µM, and p38 inhibitor (SB203580) was used at a concentration of 100 µM. It should be underlined that the concentrations of all applied inhibitors were chosen in the preliminary experiments, and neither of inhibitors affected mast cell viability, as examined by staining with trypan blue.In independent experiments, mast cells were pretreated with various signaling pathway inhibitors for 1 h at 37°C in a water bath with constant stirring, before main procedure accomplishments . Src kinase inhibitor (Src I-1) was used at a concentration of 0.01 µM, PLC/PLAAfter incubation with 5 µg/mL of IgE alone for either 0, 5, 15, 30, 60 or 120 min, purified mast cells were washed with PBS and lysed in RIPA buffer on ice for 20 min, sonicated and centrifuged . Total protein of the cell lysate was assessed by BCA reagent. Cell lysates containing 50 µg of crude cell lysate protein were analyzed by 10% SDS-PAGE followed by immunoblotting. Protein expression was detected using anti-phospho-ERK1/2 (Thr-202/Tyr-204), anti-phospho-p38 (Tyr-182) or anti-phospho-PI3K (p85 Tyr458/p55 Tyr199) antibodies, and appropriate antibodies that recognize these MAP kinases or PI3K, irrespective of their phosphorylation states. HRP-conjugated anti-mouse, anti-goat or anti-rabbit IgGs were used as secondary antibodies and then visualized using chemiluminescence reagents.6 cells/mL, were incubated with IgE at 1 µg/mL or 5 µg/mL or without IgE (native mast cells) for 1, 6 or 24 h at 37°C in a humidified atmosphere with 5% CO2. After incubation, cells were washed with cDMEM in order to remove unbound IgE, fixed using 1% CellFIX solution for 15 min at 4°C, washed twice, and finally resuspended in 1% PBS. For the measurement of FcεRI expression, mast cells were initially incubated with mouse anti-rat FcεRI IgG1 antibodies or mouse IgG1 isotype control with irrelevant specificity  at 4°C for 45 min. After washing with 1% PBS, cells were stained with FITC-conjugated goat anti-mouse IgG1 antibodies at a final concentration of 5 µg/mL at 4°C for 45 min in the dark. Following this, the cells were washed twice and finally resuspended in 300 µL of 1% PBS prior to fluorescence assessment. Ten thousand events in each sample were analyzed using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences). IgE-dependent mast cell FcεRI expression was presented as a percentage of FcεRI MFI (mean fluorescence intensity) measured in native mast cells (referred to as 100%). After each period of incubation mast cell viability was examined using the trypan blue exclusion test.Purified mast cells, at a concentration of 2×10Results are expressed as mean value ± standard error of the mean (SEM) and tested statistically by Student's t-test for “small groups”. Results were considered statistically significant when p<0.05.The effects of various concentrations of IgE, from 0.1 µg/mL to 5 µg/mL, on mast cell degranulation and preformed mediator release were evaluated first. We found that IgE alone, used at concentrations of 0.1, 0.5, 1, 1.5, and 3 µg/mL, failed to induce detectable mast cell degranulation and histamine release. However, it was noted that when stimulated with 5 µg/mL of IgE for 1 h, mast cells were activated to secrete histamine. The level of 5 µg/mL IgE-induced histamine release was significantly (p<0.01) higher than in control but statistically (p<0.05) lower than anti-IgE-challenged histamine secretion . Time-code novo TNF production. As shown in de novo TNF synthesis, the expression of TNF mRNA was analyzed. qRT-PCR was carried out and the relative expression of TNF mRNA in IgE-stimulated mast cells (5 µg/mL) compared to non-stimulated cells was assessed. It was revealed that IgE alone was capable of elevating TNF mRNA expression in mast cells in a time-dependent manner . ERK inhibitor and Src kinase inhibitor almost completely abolished, and PLC/PLA2 inhibitor significantly decreased, cysLT generation mediated by IgE alone. Mast cell pretreatment with Src I-1 or SB203580 resulted in a statistically significant (p<0.001) inhibition of IgE-induced TNF production and release. By contrast, ERK inhibitor was found to slightly attenuate IgE-mediated TNF synthesis . The same inhibitor did not affect spontaneous mast cell migration. Src inhibitor and PI3K inhibitor both caused significant  reduction in CCL5-induced, TNF-induced as well as in spontaneous migration of IgE-coated mast cells  and PI3K upon stimulation with IgE alone. As can be observed in In the current study, the FcεRI expression was evaluated on native mast cells as well as on mast cells exposed to IgE used at concentrations of 1 µg/mL and 5 µg/mL for 1, 6 and 24 h. Flow cytometry analysis revealed that native mast cells expressed surface FcεRI receptor. We found that the baseline level of FcεRI expression was significantly up-regulated (p<0.05) upon 1 h incubation with IgE at 5 µg/mL, and reached 163.6±44.3% of control FcεRI expression on native mast cells. The treatment of mast cells with 5 µg/mL of IgE following a 6 h incubation resulted in a statistically significant (p<0.05) increase in FcεRI level, compared with the control unstimulated mast cells. Interestingly, the FcεRI expression on mast cells after prolonged exposure (24 h) to IgE at 5 µg/mL was comparable with control expression. The exposure of mast cells to 1 µg/mL of IgE did not cause alteration in surface FcεRI level, at any time point examined , Fig. 7Bde novo synthesis and release of eicosanoids, cytokines and chemokines The central dogma of mast cell biology holds that FcεRI cross-linking by specific IgE and antigen initiates signaling cascade activation, leading not only to immediate preformed mediator secretion, but also to de novo TNF synthesis and release de novo TNF generation than anti-IgE stimulation at both TNF mRNA and TNF released levels.In our study we also stated that IgE alone directly triggered mature rat mast cells to preformed mediator secretion, as assessed by histamine release, and cysLT production. Importantly, the release of mediators was substantial only when IgE at 5 µg/mL was employed. So far, only a handful of studies have examined the direct effect of IgE on mast cell degranulation and eicosanoid generation, and the results remain ambiguous. Some earlier studies have shown that IgE by itself induced BMMCs and human lung mast cells (HLMCs) degranulation, however only when mast cells were stimulated with at least 3 µg/mL of IgE Under physiological conditions, the mast cell number in tissues is high and relatively constant de novo-synthesized TNF. The Src kinase-initiated signal also appeared to be vital for IgE-affected rat peritoneal mast cell increased migration towards CCL5 and TNF. Interestingly, spontaneous migratory response of IgE-coated mast cells was only partially abrogated through the withdrawal of Src kinases signal.The precise signaling mechanisms underlying mast cell response mediated by IgE alone have yet to be clarified. It is highly likely, however, that IgE-dependent signaling events are similar, if not the same, to the classical pathway cascade elicited by FcεRI aggregation 2+ in a PLC-dependent fashion 2 and ERK i.e. signaling molecules deeply involved in arachidonic acid metabolism de novo TNF generation are consistent with these observations. We also documented that IgE-affected spontaneous migratory response was independent of PLC/PLA2, and was only partially generated through the PI3K-involved pathway. Alternatively, the effect of IgE alone on the CCL5- and TNF-induced migration of mast cells was mediated by PI3K and to some extent by PLC/PLA2. These discrepancies in the mechanistic basis between spontaneous and induced migratory responses of IgE-coated mast cells may be a result of additional/common pathways triggered via chemoattractant receptors.It was established that IgE alone triggered the phosphorylation of PKB  and several MAP family kinases, i.e. ERK, p38, JUN N-terminal kinase (JNK) in vivo, and so mast cell FcεRI occupation with IgE can be relatively high. Under these conditions, IgE per se may act as a mast cell activating factor. Moreover, since it is well-known that FcεRI is present not only on mast cells but also on basophils, eosinophils, neutrophils, monocytes, and on different subsets of dendritic cells In conclusion, our results, taken in concert with previous data, clearly indicated that IgE by itself influences mast cell activity and releasability. It should be emphasized, however, that IgE alone effectively influences mast cell biology only when used at higher concentrations. Of note, there are some circumstances when IgE level is elevated


EHP explores the current state of knowledge about outdoor SHS exposure.Inhaling secondhand cigarette smoke (SHS), also known as passive smoking, can cause cancer and respiratory and cardiovascular disease.,“Outdoor SHS is an emerging topic in the tobacco control community,” says review coauthor Esteve Fernández, an epidemiologist at the Institut Català d’Oncologia–ICO in Barcelona, Spain. Surveys indicate that public support for banning outdoor smoking has increased in recent years,For the current review Fernández and colleagues analyzed data from 18 scientific papers published between 2005 and 2012, which measured SHS exposure at outdoor settings in Europe, the United States, Canada, Australia, and New Zealand. Sites included hospitality venues , airports, parks, streets, entrances to buildings, and college campuses.2.5). Measured average levels of PM2.5 ranged from 8.32 μg/m3 to 124 μg/m3 at outdoor hospitality venues where smokers were present, and from 4.6 μg/m3 to 17.8 μg/m3 at other outdoor settings. Individual point measurements exceeded 1,000 μg/m3 in some cases. Densely packed smokers, partially enclosed outdoor areas, low wind speeds, and closeness to people smoking all contributed to high levels of outdoor SHS. Smoke-free indoor settings near outdoor smoking areas also had elevated PM2.5 levels, with mean concentrations ranging from 4 μg/m3 to 120.51 μg/m3.In most of the studies reviewed, the main marker for SHS was fine particulate matter  better reflect personal exposure to SHS.The compiled data also highlight the need for better standardized methods in future studies. PMSHS contains more than 7,000 chemicals, including about 70 known and probable carcinogens, as well as toxicants and irritants.


Using the patch-clamp technique, we examined the functional expression of BK channels in neurons of the SNr and showed that the channels were activated or inhibited by internal high-energy phosphates (IHEPs) at positive and negative membrane potentials, respectively. SNr neurons showed membrane potential hyperpolarization under glucose-deprivation conditions which was attenuated by paxilline, a specific BK channel blocker. In addition, Fluo-3 fluorescence recording detected an increase in the level of internal free calcium ([Ca2+]i) during ischemic hyperpolarization. These results confirm that BK channels are present in SNr neurons and indicate that their unique IHEP sensitivity is requisite in neuronal ischemic responses. Bearing in mind that the KATP channel blocker tolbutamide also attenuated the hyperpolarization, we suggest that BK channels may play a protective role in the basal ganglia by modulating the excitability of SNr neurons along with KATP channels under ischemic stresses.Whether large conductance Ca BK channels are thought to be present and functional in various neurons of the central nervous system. They contribute to action potential repolarization in neostriatal cholinergic interneurons BK channels play important roles by couple multiple factors, such as intracellular CaThe response of BK channels to intracellular phosphates determines their involvement in the function of neurons and varies across different neurons. One type of potassium channel in substantia nigra pars compacta (SNc) neurons with a high conductance of 220 pS is activated by oxygen deprivation and inhibited by internal ATP, ADP, and AMP-PNP  at positive membrane potential The SNr, the output nuclei of the basal ganglia, is the central gate controlling the propagation of seizures, and inactivity of its GABAergic neurons, the major neurons of the SNr, is believed to protect animals from generalized seizures caused by metabolic stresses such as hypoxia and hypoglycemia Slo gene, has been cloned from a cDNA library of human substantia nigra by hybridization screening Slo1 mRNA has not been detected in rat SNr Slo1 mRNA in brain regions such as the ventromedial hypothalamic nucleus neurons Existing molecular biological evidence for the expression of BK channels in the SNr is inconclusive. Positive evidence suggesting their existence includes immunoreactivity of the SNr for the α subunit (Slo1) To understand the alteration of neuronal activity in the SNr under different metabolic conditions, i.e. ischemic conditions, we used cell physiological techniques and methods to investigate whether BK channels are functionally expressed in SNr GABAergic neurons. We found that BK channels are present in SNr neurons, are bidirectionally modulated by IHEPs, and are involved in the hyperpolarization of plasma membrane potential under glucose-deprived conditions.Ih) at outputs of transmembrane potentials more positive than −120 mV  from C57BL/6 mice, with a longitudinal diameter of 19.4±0.2 µm, membrane potential of −51.8±0.4 mV, firing rate of 10.4±0.4 Hz and firing half-width of 3.4±0.1 ms, were recorded in perforated whole-cell recording mode. Thirty-eight of the 294 neurons were stimulated by serially-stepped current pulses, and did not show hyperpolarization-activated currents  of the channel was dependent on [Ca2+]i at both positive and negative transmembrane potential . According to the gradient of the I–V curve, the conductance of the BK channel was 301 pS  between −60 ∼ +60 mV  values of 270 µM (n = 9–12) and 12 µM (n = 6–9) were obtained at transmembrane potentials of −60 mV and +60 mV respectively by fitting the Hill equation to Popen-[Ca2+]i. When [Ca2+]i was limited to not more than 1 mM, the conductance of the channel did not change. We observed that BK channel conductance decreased by 15% (n = 7) and 42% (n = 3) at +60 mV, and decreased by 0% and 18% (n = 3) at −60 mV, at extremely high [Ca2+]i of 3 mM and 10 mM, respectively   , and theependent . This deATP channels, did not affect BK channel currents when the channels were opened by 100 µM Ca2+ at 0 mM ATP (8 patches) , a specific blocker of β-cell Kpatches) . This rePopen-ATP data at −60 mV, gave a Hill coefficient (n) of −1.68 and an 50IC  of 0.65 mM (n = 15) (n = 9), but 3 mM AMP did not have a visible effect on BK channel currents (n = 4). The conductance of BK channels was independent of internal phosphates at both positive and negative transmembrane potentials. These results suggest that IHEPs bidirectionally modulate the opening of the BK channels of SNr neurons.To observe the effect of IHEPs on BK channels, phosphates were added to the inside of excised patches. ATP decreased the open probability of BK channels at negative potential in a dose-dependent manner, but had no significant effect at positive potential . Fitting(n = 15) . The effP<0.0005), respectively, 9.7±0.8 min and 12.2±1.9 min (P>0.05) after glucose-deprivation began. Tolbutamide was used to test for hyperpolarization-accompanied reductions in intracellular ATP, since pancreatic β-cell KATP channels are rapidly sensitive to intracellular ATP and are highly expressed in SNr neurons We examined the effect of glucose deprivation on changes in membrane potential in SNr neurons. The 159 SNr neurons exposed to glucose-free ACSF  could be divided into three groups based on changes in membrane potential: 17 (11%) were depolarized until the membrane potential disappeared, 28 (18%) were hyperpolarized after a slight depolarization, and 114 (71%) were directly hyperpolarized. Initial characteristics of these three response types are detailed in n = 13) were subjected to glucose deprivation treatment and membrane potential and fluorescence were co-recorded. A perforated whole-cell patch-clamp configuration was successfully maintained in only four of the neurons (P>0.05). The gray value increased by 32% (P<0.05) at max hyperpolarization (14.6±2.0 mV) relative to before the onset of glucose deprivation treatment. From these results we hypothesize that intracellular free calcium may contribute to the hyperpolarization caused by glucose deprivation injury by regulating BK channels.To examine the involvement of intracellular free calcium in the cellular function of BK channels during glucose deprivation, experiments were performed in Fluo3-AM-loaded SNr neurons. Neurons  and the half-width by 61% , and decreased the firing rate by 33% . Under glucose-deprivation conditions, paxilline attenuated the hyperpolarization in 10 out of 12 neurons. Charybdotoxin (4/5), a blocker of IK and BK channels, and TEA (44/49) attenuated the hyperpolarization to a similar extent as paxilline. Apamin, a small potassium channel blocker, did not affect the hyperpolarization (4/4). These results, in addition to our finding that the open probability of the channels is not higher than 17% when [Ca2+]i is limited to the physiologically-relevant level of 1 µM . 2 mM TEA increased the peak amplitude by 37% . Here, TEA was used instead of paxilline, since it is easier to wash out from plasma membranes. The neurons had an initial resting potential of −48.9±0.8 mV and showed max hyperpolarization of 13.9±1.3 mV at 14.1±1.7 min after the onset of glucose deprivation. TEA and tolbutamide markedly attenuated hyperpolarization in 43/48 and 46/48 of neurons, respectively. The earliest attenuations caused by TEA were observed on average at 19.5±1.8 min of glucose deprivation, significantly later than max hyperpolarization (P<0.05), while attenuations caused by tolbutamide were observed from the onset of hyperpolarization. The effect of TEA did not decrease as glucose deprivation was prolonged, while tolbutamide lost its effect in 16/21 neurons within 60 min of glucose deprivation, on average at 24.8±3.4 min (n = 16) after the onset of glucose deprivation. When the first effects of TEA occurred, the membrane potential was −59.3±1.5 mV (n = 43), more positive than that at max hyperpolarization  and more negative than that at resting potential . These results suggest that BK channels collaborate with KATP channels in the maintenance of glucose deprivation-induced hyperpolarization of plasma membrane potential; BK channels open after KATP channels and remain functional under conditions when KATP channels are no longer functional.To discriminate the function of BK channels from that of Kpeatedly , after t2+ and contribute to membrane potential hyperpolarization. The results presented here provide insights into how BK channels regulate SNr GABAergic neuron excitability by their unique response to intracellular IHEPs.We report here that functional BK channels are present in the cell body of SNr GABAergic neurons. These channels are modulated bidirectionally by IHEPs at positive and negative transmembrane potentials. Under glucose-deprived conditions BK channels are activated by a decrease in intracellular ATP and an increase in intracellular Ca2+, should be able to chelate [Ca2+]i to 34 µM and 13 µM, respectively, as calculated using Maxchelator (http://www.stanford.edu/~cpatton/maxc.html). At these calculated concentrations of free calcium, Popen should be 18% or 6% at −60 mV and 82% or 60% at +60 mV, respectively, according to the Popen-[Ca2+]i curve  or ischemic (n = 4) conditions in SNr neurons (data not shown). Moreover, BK channels of SNr neurons and LGP neurons These effects of IHEPs may reflect differences in molecular structure between BK channels of the basal ganglia and other BK channels in the central nervous system. BK channels of the three basal ganglial nuclei, SNr, SNc, and LGP, are inhibited by IHEPs at negative-membrane potential 2+]i sensitivity. The EC50 of [Ca2+]i for SNr BK channels was 12 µM at a membrane potential of +60 mV, larger than that for BK channels in the SNc and LGP, while at a membrane potential of −60 mV, the EC50 of [Ca2+]i for SNr BK channels was 270 µM, lower than that for BK channels in the SNc and LGP. In addition, channel conductance was almost the same for inward and outward currents in the SNr BK channels, while BK channels in the SNc and LGP showed slight inward rectification. It is worth noting that the [Ca2+]i and voltage dependence of BK channels in the SNr are lower and conductance is larger than in BK channels formed by slo 1 and β4 subunits Here, differences between BK channels in SNr neurons and those in the SNc and LGP were mainly in their [CaTaken together, these results indicate that functional BK channels, whose molecular structure may differ from BK channels in other brain regions, are present in basal ganglia; differences in the pattern of regulation between basal ganglial BK channels and BK channels in other brain regions suggest that modulation of basal ganglia output by the selective regulation of BK channels in basal ganglial neurons may be possible.ATP channels (25/34) obtained from mutant mice which lacked the Kir6.2 subunit of the KATP channel ATP channel in initiating the hyperpolarization induced by glucose deprivation. In a previous report, we showed that the KATP channel of SNr neurons is opened by ATP depletion induced by oxygen-deprivation treatment, and plays a protective role in hypoxia-induced generalized seizure It is interesting that BK channels are involved in the maintenance of hyperpolarization induced by glucose deprivation. The pathology of SNr has been highlighted in recent reports describing functional or histological injury in patients with basal ganglial diseases. Excessive activation of GABAergic output from the SNr in Parkinsonism is induced by reduced dopamine input to the striatum from the SNc 2S2O4, CCCP, tolbutamide and tetraethylammonium (TEA), Flou-3 AM, and MTT were purchased from Sigma, USA. ADP and AMP were purchased from Amresco (USA). L-cysteine hydrochloride monohydrate was purchased from Wako (Japan). Deionized water (18 MΩ/cm) was obtained using a MILLIPORE-Q-B device  and used for all solutions.Papain, kynurenic acid, paxilline, trypsin, nystatin, Tris, ATP, GTP, NaC57BL/6 mice were obtained from the Animal Experiment Center of Peking University Health Science Center (PR China). Mice lacking the Kir6.2 subunit of KATP channels (KO mice) were kindly provided by Prof. Susumu Seino (Kobe University) in vivo and were used in MTT staining experiments  or Tris-hydroxymethylaminomethane (Tris) . ACSF was saturated with 100% O2 30 min before use until the end of experiments. Coronal slices containing the rostral region of the SNr (thickness: 300 µm for cell dissociation or 130 µm for experiments using slices) were sectioned in ACSF solution with a vibroslicer . The rostral region of the SNr was selected because it does not contain dopaminergic neurons, which are situated in the central region of the SNr Single neurons, obtained from P17-20 mice, enable rapid and complete alteration of extracellular conditions and were used in inside-out patch-clamp configuration and Fluo-3 fluorescence recording experiments. Brain slices, obtained from 13–14 week-old mice, maintained the living environment of synapses and cell bodies close to that To obtain single SNr neurons, slices were incubated in an enzyme solution containing 26 units/ml papain, 1 mM kynurenic acid, and 1.5 mM L-cysteine hydrochloride monohydrate in ACSF, for 30 min at 30°C V = 0). Fire-polished pipettes were made from borosilicate glass capillaries using a puller  and polisher . Pipettes had a resistance of 3–4.5 MΩ when filled with pipette solution containing (in mM): 140 KCl, 0.1 CaCl2, 0.2 MgCl2, 10 HEPES, pH 7.2. The appropriate amount of CaCl2 was added to the pipette solution to make the bath solution. Beakers and tubes with good chemical resistance were used and 1 mM EGTA was used to remove the remaining Ca2+ in the perfusion tubes before measurement of calcium-dependence I = 0) in isolated single cells. Pipettes had a resistance of 2–3 MΩ when filled with solution containing (in mM): 140 KCl, 10 HEPES, 50 µg/ml nystatin, pH 7.2. The bath solution used ACSF, or glucose/oxygen free ACSF. Bath solutions were perfused at 4–6 ml/min. Serial resistance and membrane capacitance were compensated as much as possible by the amplifier without producing an oscillation in the potential signal. Data were analyzed with Clampfit 9.0.We obtained all data using an Axonpatch 200B patch-clamp amplifier (whose strength lies in single channel recording), and Digidata 1322 , and data were analyzed with pCLAMP 9.0 (Axon Instruments). A low-pass filter of 2 kHz and a sampling frequency of 10 kHz were used. Single BK channel currents were recorded using an inside-out configuration, which enables rapid and complete alteration of the internal solution of the plasma membrane and is convenient for the investigation on the responses of ion channels to intracellular factors, in voltage-clamp mode  solution containing (mM): 145 NaCl, 4 KCl, 1 MgCl2+ fluorescence was excited by a wavelength of 488 nm from a mercury lamp and was collected using a 520 nm filter Recovered cells were incubated for 20–30 min in ACSF containing 0.5–1 µM Fluo-3 acetoxymethyl ester (AM) in a culture dish placed in a container filled with oxygen and 100% humidity. Fluo-3 AM was dissolved in dimethyl sulfoxide to make a stock solution before diluting with ACSF, maintaining the final dimethyl sulfoxide concentration lower than 1‰. Cells were rehabilitated by ACSF perfusion for at least 30 min and then used for experiments. Cat-test. Differences were considered significant if P<0.05.Data are presented as means ± standard error. The significance of differences between groups was calculated using the Student’s Figure S1Characteristics of dissociated SNr neurons during glucose deprivation treatment. (A–C) Continuous recording of membrane potential in wild type  and knock-out  neurons. The responses to glucose deprivation in (A), (B), and (C) are type 2 (directly hyperpolarized), type 3 (hyperpolarized after a slight depolarization), and type 2 responses, respectively. (D) Representative pattern of membrane-potential responses to serially-stepped pulses. Traces correspond to the section in (A) marked by “mminV) corresponds to the maximum hyperpolarization and that the maximum membrane potential (mmaxV) corresponds to the maximum depolarization. (G) Histogram showing the time from the start of the glucose deprivation treatment (t = 0) to mminV and mmaxV. *, P<0.05; **, P<0.01; ***, P<0.005; ****, P<0.001; *****, P<0.0001. All 159 WT neurons had an average longitudinal cell body diameter, resting potential, firing rate, and half-width of about 19.2±0.4 µm, −52.9±0.5 mV, 10.8±0.6 Hz, and 3.1±0.1 ms, respectively, and the 34 KO neurons had an average longitudinal cell body diameter, resting potential, firing rate, and half-width of about 20.1±1.2 µm, −50.0±0.9 mV, 10.3±1.7 Hz, and 4.2±0.3 ms, respectively. P values for the comparison of these characteristics between WT and KO neurons were determined using the Student’s t-test and were 0.175, 0.0071, 0.4188, and 0.0018, respectively. These electrophysiological characters of WT and KO neurons are similar to results from our previous report (TIF)Click here for additional data file.Figure S2The effect of 2 mM TEA on the substantia nigra (SN). Serial coronal slices (130 µm) containing the SN, indicated by closed curves, from C57BL/6 mice (d15-20) were prepared using a vibroslicer  in ice-cold, O2-saturated artificial cerebrospinal fluid (ACSF) containing the following (in mM): 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4 (Tris). Slices were then incubated for 30 min in ASCF in multiple perfusion chambers at room temperature (23–24°C) for recovery. Each 1.5 ml chamber had a perfusion rate of 4.0±0.2 ml/min and contained 1–2 slices. Here, the environment of the tissue could be regulated by exchanging the perfusion solution. The perfusion solution was changed to ACSF alone, ACSF without glucose or without glucose and oxygen, or to these solutions containing 2 mM TEA. Tissues were then incubated in MTT solution (i.e. 0.5 mg/ml MTT dissolved in ACSF) for 20 min followed by fixing with 4% paraformaldehyde for 2 hours, washing in phosphate buffer, mounting on a glass slide, and drying overnight **, P<0.01; *****, P<0.0001.(TIF)Click here for additional data file.