The goal of this study was to examine how a chronic Na+ channel leak current in developing Purkinje cells in the heterozygous Lurcher mutant (+/Lc) affects the expression and distribution of the α3 subunit of the Na+/K+ ATPase pump, a key component of the homeostasis system that maintains ionic equilibrium in neurons. The expression pattern of the catalytic α3 Na+/K+ ATPase subunit was analyzed by immunohistochemistry, histochemistry, and Western Blots in wild type (WT) and +/Lc cerebella at postnatal days P10, P15, and P25 to determine if there are changes in the distribution of active Na+/K+ ATPase subunits in degenerating Purkinje cells. The results suggest that the expression of the catalytic α3 subunit is altered in chronically depolarized +/Lc Purkinje cells, although the density of active Na+/K+ ATPase pumps is not significantly altered compared with WT in the cerebellar cortex at P15, and then declines from P15 to P25 in the +/Lc cerebellum as the +/Lc Purkinje cells degenerate.A common assumption of excitotoxic mechanisms in the nervous system is that the ionic imbalance resulting from overstimulation of glutamate receptors and increased Na The mature pump resides in the plasma membrane and exports 3 Na+ ions for every two K+ ions it imports at a cost of 1 ATP molecule, resulting in a net outward, hyperpolarizing current [α) and a beta (β) unit, though in some cell types the pump contains an additional FXYD protein [α subunit is the catalytic unit and is responsible for binding of Na+ and K+ ions, ATP, and ouabain (an inhibitor). The β subunit plays a crucial role in the structure and maturation of the Na/K pump, including, for example, aiding in the tracking of the α subunit from the ER to the plasma membrane [α3 and β1 subunits, but some other cerebellar neurons  or structures  also express the α3 subunit [The Na current , 3. All  protein , 5. Thermembrane . Cerebel subunit . The FXY subunit , but thi+ and Ca++ influx that overwhelms cellular energy metabolic systems leading to cell death [+ levels to help restore cellular homeostasis, but the increased demand for ATP could be deleterious. Alternatively, decreased Na/K pump activity in injured cells may allow for increased intracellular Na+, but the decreased demand for ATP from less active Na/K pumps may be neuroprotective.Na/K ATPase activity in neurons may contribute as much as 240 pA to the resting membrane potential and pump activity is increased as a function of neuronal activity . Changesll death , 18. Botδ2 glutamate receptors (GluRδ2) with the Lurcher mutation, GluRδ2Lc, found that ectopic expression of the mutant receptor in cultured cells causes a decline in ATP levels [Lc mutation in GluRδ2 turns the receptor into a constitutively open membrane channel that chronically depolarizes the cells expressing the mutant receptor [in vitro studies, the authors speculate that over activation of the Na/K pump in response to the chronic Na+ leak may consume cellular ATP levels leading to cell death [+ leak current mediated by the mutant GluRδ2Lc receptor in developing Purkinje cells in the heterozygous Lurcher (+/Lc) mouse mutant affects the cellular distribution of α3 subunits and active Na/K pumps.A recent study of HEK293 cells expressing P levels . The Lc receptor . In the ll death , 21. Theδ2 receptor was first identified based on its homology to NMDA and AMPA glutamate receptors, but the receptor does not bind to most glutamate agonists or antagonists, nor does it appear to carry a membrane current under normal circumstances. GluRδ2 is preferentially expressed in cerebellar Purkinje cells, and in the +/Lc mutant, cerebellar Purkinje cells become chronically depolarized at the end of the first postnatal week when GluRδ2 receptors are inserted at Purkinje cell-parallel fiber synapses. The majority of +/Lc Purkinje cells degenerate during the first month of postnatal life starting at the end of the first week via pathways that have been described as either apoptotic, autophagic, or necrotic [Lc cerebellum [Lc Purkinje cells, the chronic depolarization caused by the constitutive cation leak current from the GluRδ2Lc channel stresses the neuron's ion exchange and energy production systems [Lc Purkinje cells through P25 [Lc Purkinje cells in vitro [Lc Purkinje cells contain numerous dilated mitochondria, suggesting mitochondrial dysfunction [Lc Purkinje cells also contain significantly higher levels of nitric oxide synthase (NOS) activity and protein nitration [++ will stimulate NO production. NO and O2− free radicals will react to form peroxynitrite, a highly reactive oxidizing agent which can damage and kill cells by tyrosine nitration of critical proteins and membrane lipid peroxidation [α3 subunit is increased, or at least more concentrated, in individual +/Lc Purkinje cells through P25. However, as +/Lc Purkinje cells degenerate by P25, total expression levels of α3 and its 40 kD breakdown product in the +/Lc cerebellum declines compared with WT cerebella. The density of active Na/K pumps assayed ex vivo is not altered at P15, near the peak of +/Lc Purkinje cell death, and decreased at P25 when there are relatively few remaining +/Lc Purkinje cells. Our interpretation of the results is that while the expression of the α3 subunit may be increased within individual +/Lc Purkinje cells in response to the chronic stress, potential increases in overall ATPase pump activity and ATP consumption due to increased expression of the α3 subunit may be limited by the deactivation and/or degradation of many α3 subunits.The gene for the GluRnecrotic , 22, 23.rebellum . We have systems , 26. In in vitro . In addifunction . +/Lc Puxidation , 30. Theδ2Lc+/ mutant and GluRδ2+/+ wild type (WT) pups were generated by mating B6CBACa Aw-J/A-Grid2Lc/J males with WT females (C57BL/6J), both from Jackson Laboratories. All animals were housed in standard conditions  in the animal facilities at the Maryland Psychiatric Research Center and provided with food and water ad libitum. Males were harem mated with one male per two females. The day of birth was counted as postnatal day 0 (P0). The animal facilities are fully accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC) and the studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals provided by the NIH.GluRμg/g) or by decapitation. Following the perfusions with 4% paraformaldehyde, brains were removed from the skull, postfixed for 2 hours, and then cryoprotected with 20% sucrose in 10 mM phosphate buffed saline (PBS). At least 48 hours later, the fixed brains were embedded in OCT and frozen in isopentane. Following decapitation, freshly dissected brains were either bisected and frozen in aluminum foil for Na/K pump activity assays or the cerebellum was isolated and frozen in crushed dry ice for Western blot analysis.Mice were either euthanized by cardiac perfusion with 0.9% saline followed by 4% paraformaldehyde  or rabbit polyclonal anti-α3 Na/K ATPase  or rabbit polyclonal anti-activated caspase-3  and monoclonal mouse anti-α3 Na+/K+ ATPase . The sections were rinsed 3 times in PBS and then incubated for 2 hours with fluorescent-labeled secondary antibodies . After incubation, they were rinsed once in 10 mM PBS and incubated with 300 nM DAPI, then rinsed 3 times in 10 mM PBS, once in distilled water, and coverslipped with gel mount. The finished slides were then photographed using either an Olympus FV500 laser scanning confocal microscope or a Zeiss Axioplan fluorescence microscope. Confocal digital images were cropped and adjusted using Adobe Photoshop for color balance and intensity. All immunofluorescence experiments included slides (WT and +/Lc) with no 1antibody incubation as a control for nonspecific immunolabeling. The specificity of both the mouse monoclonal (ABR Affinity BioReagents) and the rabbit polyclonal (Upstate/Millipore) anti-α3 antibodies was also tested with Western blots using homogenized cerebellar tissue from WT and +/Lc mutant mice (data not shown). The ABR mouse monoclonal antibody labeled a single 110 kD band that represents the unbound, native α3 isoform, while the Upstate rabbit polyclonal antibody labeled two bands, the 110 kD α3 isoform and a 40 kD band that has been shown to be a byproduct of α3 isoform cleavage by calpain activity [Fixed, frozen brains were cut at 12 activity .Lc cerebella, digital images of α3 isoform and calbindin immunolabeling of Purkinje cells were taken at 40x on a Zeiss Axioplan with an Olympus DP70 CCD camera within the first two days after staining all of the sections (to avoid uneven fading artifacts). The raw, unprocessed images were then analyzed using Metamorph Version 7.0r1. Threshold and colocalization functions were used to selectively measure the intensity of α3 isoform immunolabeling in areas that colocalized with calbindin-stained Purkinje cells in the same section. At least 3 molecular layer regions per cerebellum were selected from vermal sections in each WT or +/Lc cerebella and the mean value calculated for each cerebellum. The number of WT and +/Lc cerebella analyzed at each time point was P10, n = 5 WT, 4 +/Lc; P15, n = 5 WT, 5 +/Lc; P25, n = 4 WT, 5 +/Lc. The intensity of the α3 isoform fluorescence signal at P10, P15, and P25 is expressed as a percent change from labeling intensity in control Purkinje cells at P10.For the semiquantitative comparison of Na/K ATPase immunolabeling in WT and +/Lc cerebella were collected from freshly dissected brains, rapidly frozen in dry ice and stored at −70°C until processed. Each cerebellum was homogenized in a buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1%SDS, 10 μL/mL Protease inhibitor cocktail (Sigma), 1 mM PMSF, and 1 mM NaVO4. The homogenate was centrifuged at 15,000 rpm for 15 minutes. Protein concentration in the supernatant was measured using a BioRad protein assay kit. Protein extracts were diluted in Laemmli sample buffer with β-mercaptoethanol and denatured at 37°C for 5 min and 10 μg of protein per well was resolved on a Tris-glycine gel. Protein was transferred overnight at 4°C onto a PVDF membrane. The membrane was rinsed with 5% nonfat dry milk dissolved in 1 × TBS. It was then incubated in the anti-α3 Na/K ATPase , diluted in TBS/0.1% Tween (TBS-T) with 1% milk overnight at 4°C, and then rinsed 3 × 10 min each with PBS-T. The sample membrane was incubated in alkaline phosphatase-conjugated secondary antibody (diluted 1/1000) and the protein was detected using Bio-Rad immun-Star chemiluminescence kit. Film exposed to the chemiluminescent signal was digitized using a light box and Pixera Pro150ES digital camera connected to a Power Macintosh. The optical density of the images was calibrated using a photographic calibration step tablet (Kodak) so that data is collected in the linear range of the O.D. The relative density of the labeled protein bands was determined using densitometric measurements with ImageJ. In all studies, once data from the antigen of interest had been collected, the membrane was stripped and labeled for total protein with India ink as a loading control. The density of α3 isoform protein bands was corrected for the total amount of protein by calculating the ratio of the density of the α3 isoform band and the total protein. Changes in the density of the corrected α3 isoform band were expressed as a percent of the averaged density of the α3 isoform bands from P25 controls within the same gel.WT and +/Histochemistry. The density of active Na/K pumps was measured in frozen tissue sections using a procedure modified from previously published protocols [Lc brains were bisected and rapidly frozen in dry ice for 2 min. The brains were then cut the same day in parasagittal sections at 20 μm. A kidney from one of the wild type animals was also frozen and a 20 μm section of kidney was placed on each slide as a positive control. Two slides with 2 sections per slide from each brain were used in the assay. The sections were fixed for 10 min in 2% paraformaldehyde, rinsed 3 times each in PBS, rinsed twice in 50 mM Tris/100 mM sucrose (pH 7.4), and then rinsed 3 times for 15 min each time in Tris/sucrose buffer. They were then incubated for 20 min at 37°C in a lead citrate mix pH 8.8 of 4mM Potassium Citrate, 4 mM Lead Nitrate, 250 mM Glycine, 25 mM potassium hydroxide, 20% DMSO, 10 mM para-Nitrophenylphosphate (p-NPP), and 2.5 mM Levamisole. Sections were then rinsed in distilled water, rinsed for 10 min in TRIS/sucrose buffer, and rinsed again 2 times in distilled water. Sections were developed for 2 min in 1% ammonium sulfide, rinsed 2 times in distilled water, rinsed for 10 min in PBS on a rocker, rinsed in distilled water, covered in crystal mount, and allowed to dry at room temp. As an assay for ouabain insensitive activity, one slide from each brain was treated with 10 mM Ouabain during the final Tris/sucrose incubation and the lead citrate incubation.rotocols , 33. WT Quantification of Enzyme Activity. Stained slides were photographed at 5x on a Leica DMR microscope with a 24-megapixel Power Phase Scanning camera . To decrease variations in background illumination, images of both the sections and adjacent blank areas of slide were photographed. The background images were subtracted from the section images using MatLab. Because of the large number of sections to analyze in each histochemical experiment, digital images were collected over three consecutive days. To calibrate for any potential differences between images taken in different sessions , the change in average density of a selected section that was imaged all three days was subtracted from the respective day. Using ImageJ, a systematic random set of points was selected to measure optical densities. The density for the molecular layer and granular cell layers was averaged for each brain from measurements of two sections per brain per experiment. The ouabain-specific activity was calculated by subtracting the density of the ouabain treated tissue from the density of the untreated tissue.Statistical comparisons between experimental and control groups were made using two-way or one-way analysis of variance (ANOVA) and post hoc comparisons were made using Bonferroni/Dunn (Statview 5.01).α1 Na/K pump subunit is expressed in granule cells and glomeruli, α2 subunits are expressed in astrocytes, and α3 subunits are expressed in Purkinje cells, basket cell processes, and mossy fiber glomeruli [Lc mutant, the α1 and α3 subunits were initially selected for study as the most likely isoforms to show altered expression patterns. A preliminary immunohistochemical survey did not show any evidence of changes in the distribution or intensity of immunolabeling of the α1 subunit (results not shown), but there was evidence of distinct changes in the pattern of immunolabeling for the α3 subunit in the +/Lc cerebellum from P10 to P25. Vermal cerebellar sections from at least 4 WT and +/Lc cerebella at P10, P15, and P25 were double immunolabeled for calbindin and the α3 subunit using the Upstate rabbit polyclonal antibody that recognizes both the 110 kD full length α3 isoform and the 40 kD byproduct of α3 isoform cleavage in Western blots. In both WT and +/Lc cerebella from P10 to P25, the α3 subunits appear to be expressed in Purkinje cells, the molecular layer, and granule cell layer glomeruli in a similar pattern to that described by Peng et al. [α3 expression in WT Purkinje cells is diffusely distributed throughout the dendrites and cell body as shown for P15 WT Purkinje cells in Figures α3 is qualitatively different at all three ages in +/Lc Purkinje cells. From P10, the α3 isoform labeling becomes more intense and less diffuse within +/Lc Purkinje cells. As shown for P15 +/Lc Purkinje cells in Figures α3 subunit (green) is more punctate and concentrated around the primary dendrites and cell bodies so the labeling appears more intense, though there is some variation between different Purkinje cells. For example, the α3 labeling is particularly intense in the two left most +/Lc Purkinje cells in Figures α3 isoform appears to surround the stunted primary Purkinje cell dendrites especially in the lower two-thirds of the molecular layer. α3 immunolabeling is also seen around the Purkinje cell bodies but not as much within the cell body in comparison with WT Purkinje cells. α3 immunolabeling in the granule cell layer glomeruli is indicated by white asterisks in Figures α3 immunolabeling in the Purkinje cell and molecular layers in the +/Lc cerebellar cortex may be in climbing fibers, it is clear that the intensity and distribution of α3 immunolabeling is altered in the +/Lc cerebellar cortex.A previous study has shown that in an adult mouse cerebellum, the lomeruli . Since bg et al.  in the cLc Purkinje cells that appear to be in the final stages of degeneration express activated caspase-3 [α3 subunit expression persists in dying Purkinje cells, cerebellar sections from P15 and P25 +/Lc cerebella were double labeled with antibodies to activated caspase-3 and the α3 isoform and counterstained with DAPI. The distribution of α3 isoform labeling in activated caspase-3 positive +/Lc Purkinje cells was examined in cerebellar sections from three +/Lc mutants at P15. Confocal images of two such degenerating +/Lc Purkinje cells are shown in Figures Lc Purkinje cells continue to express the α3 isoform even as they degenerate. The stunted dendrites and cell body of the activated caspase-3 positive (red) +/Lc Purkinje cell shown in α3 (green and yellow) throughout the remaining dendritic tree and cell body. The +/Lc Purkinje cell shown in α3 immunolabeling. DAPI labeling (purple) in the nuclei of both activated caspase-3 positive neurons shows the condensation of the nuclei in the degenerating +/Lc Purkinje cells (white arrows). Nuclear DAPI labeling is found in the neighboring +/Lc Purkinje cells that have not yet begun to express activated caspase-3 (white arrowheads).+/aspase-3 , 35. To α3 isoform suggest that it is diffusely distributed throughout the Purkinje cell dendritic tree from P10 to P25 in the WT cerebellum. In contrast, in +/Lc Purkinje cells, α3 immunolabeling becomes more intense from P10 to P25 with punctate labeling concentrating along the Purkinje cell primary dendrites and cell bodies. As an assay for developmental changes in α3 immunolabeling, the intensity of the α3 (green) fluorescence signal coincident with calbindin labeling in WT and +/Lc Purkinje cells was measured using MetaMorph , but the age x genotype interaction does not reach significance . Post hoc analyses of the complete data set suggest that there are significant increases in α3 fluorescence intensity between P10 and P15 and between P10 and P25 and an overall significant difference between fluorescence intensity in +/Lc and WT Purkinje cells . A further exploratory post hoc one-way ANOVA analysis separating the data set by age suggests that while differences between α3 isoform immunofluorescence in +/Lc and WT Purkinje cells at P10 and P15 may contribute to the main effect, they are not significantly different when analyzed separately . However, there is a significant difference between the intensity of α3 isoform immunolabeling in +/Lc and WT Purkinje cells at P25 with higher levels in the +/Lc Purkinje cells .The qualitative descriptions of the developmental changes in immunolabeling for the etaMorph . Two-wayα3 immunofluorescence in +/Lc Purkinje cells from P10–P25 may be due to a variety of reasons, including either a redistribution of existing α3 isoforms or an increase in the expression levels of the α3 isoform. To distinguish between these possibilities, protein extracts of cerebellar tissue were prepared from WT and +/Lc cerebella at P10, P15, and P25 and the relative levels of α3 isoform expression were quantified by Western blot , but there are no significant effects of genotype  or age x genotype interactions . Post hoc exploratory analyses indicate that in the +/Lc and WT cerebella, α3 isoform protein levels at P25 are significantly higher than at P10 and P15 . A separate one-way ANOVA analysis of the data separated by age indicates that α3 isoform protein levels are only significantly different between WT and +/Lc cerebella at P25 . The results indicate that α3 isoform levels in the WT and +/Lc cerebellum increase with age after P15, but by P25 the increase in α3 levels in +/Lc cerebella is not as great as in WT cerebella.Quantitative changes in the relative optical densities of the 110 kD α3 isoform protein levels , genotype , and age x genotype interactions . Post hoc analyses indicate that group levels for the α3 40 kD fragment are significantly higher at P25 compared with P10 levels  but not between P25 and P15 or P15 and P10 . One-way ANOVA separated by age suggests that the genotype effect is primarily due to a significant difference between the amount of α3 40 kD fragment at P25 , with significantly lower levels in +/Lc cerebella. There are no significant differences between levels of the 40 kD fragment at P10 or P15. The results suggest that by P25 there is a dramatic increase in the 40 kD α3 fragment in WT cerebella, but this age-related increase is not matched in the +/Lc cerebella.Two-way ANOVA of the 40 kD n levels  indicateδ2Lc channel mediates an Na+ leak current we hypothesized that the density of active Na+/K+ ATPase pumps would be increased from P10 through P25 in +/Lc Purkinje cells in response to increased intracellular Na+ levels. Furthermore, the changes in the distribution of the catalytic α3 subunit of the Na/K pump also suggest that the distribution and density of pump activity would be altered in +/Lc Purkinje cells. To assay changes in the distribution of active Na/K pumps in the +/Lc cerebellum, p-NPP was used as a substrate in an in situ histochemical assay to detect ouabain-sensitive, potassium-dependent activity of the Na/K pump complex. Representative images of histochemical labeling for Na/K pump activity in WT and +/Lc cerebellar cortex at P15 and P25 are shown in Lc cerebella are taken at the same magnification, but the +/Lc cerebella is smaller at P15 and dramatically reduced in size by P25 compared to WT because of ongoing  +/Lc Purkinje and granule cell death. Quantitative analysis of in situ ouabain-sensitive Na/K pump activity in histological sections of wild type and +/Lc cerebellar sections at P15 and P25 shows that the overall density of active Na/K pump in the molecular and granule cell layers is within WT levels at P15  but decreases by P25 in both the +/Lc molecular layer  and granule cell layer . However, the preferential survival of +/Lc Purkinje cells in the nodulus did not appear to affect the overall decline in Na/K pump activity observed at P25 in the +/Lc cerebella in both the molecular and granule cell layers.At P15, significant numbers of +/ nodulus . To deteerebella . Followiδ2 converts this enigmatic glutamate receptor into a constitutively open cation channel that chronically depolarizes +/Lc Purkinje cells as they mature following the first week of postnatal development [Lc Purkinje cells is thought to initiate excitotoxic cell death pathways [+ and Ca++ ions places overwhelming stress on cellular energy metabolism systems [Lc Purkinje cells, in particular, mitochondrial cytochrome oxidase activity is significantly increased, possibly in response to increased cellular energy requirements [+/K+ ATPase α3 isoform expression and the density of active Na/K pumps are increased in +/Lc Purkinje cells. This increase would be consistent with an increased demand for ATP production (and increased cytochrome oxidase activity) to counterbalance the GluRδ2Lc Na+ and Ca++ leak current. However, the results indicate that while the expression of the catalytic α3 isoform may be increased in individual +/Lc Purkinje cells, the density of active Na/K pumps is not significantly increased above wild type levels in the cerebella of younger +/Lc mutants (P10 to P15). Furthermore, by 25, overall expression levels of the α3 isoform and its 40 kD breakdown product are decreased in the +/Lc cerebellum compared to WT, along with a decrease in the density of active Na/K pumps. The lower cerebellar expression levels of the α3 isoform and decreased density of active Na/K pumps by P25 in the +/Lc cerebellum may simply reflect the substantial loss of +/Lc Purkinje and granule cells by this age. While the results indicate that the density of active Na/K pump units are not significantly increased in +/Lc Purkinje cells in response to the leak current, it is important to note that in this study we have not analyzed the in vivo unit activity of Na/K pumps in living +/Lc Purkinje cells.The Lurcher mutation in GluRelopment , 22. Thepathways . One ass systems , 18. In irements . The goaα3, and β1 isoforms suggested that only the expression of the catalytic α3 subunit is altered in the +/Lc cerebellar cortex. The change in expression of the α3 isoform in +/Lc Purkinje cells was subsequently analyzed by fluorescent immunolabeling to provide information about subunit localization. The significant increase in the intensity of immunofluorescence for the α3 subunit from P10 through P25  and eventually degenerate. The Western blot data appears to favor the latter hypothesis since the only significant difference in α3 expression is a relative decrease at P25 in +/Lc cerebella when 70–90% of the +/Lc Purkinje cells and 60% of the granule cells have degenerated [Lc Purkinje cells are degenerating from at least P10 onwards with approximately 50% of the +/Lc Purkinje cells missing by P13 to P15 [Lc Purkinje cell steadily increases from P10 through P25 . In theLc cerebellar cortex is in stark contrast to the distribution of cytochrome oxidase (COX) histochemical staining at the same ages [Lc Purkinje cells at P15 and P25 stain darkly for COX, filling the entire Purkinje cell body and dendritic tree. The dramatic increase in COX activity suggests that the mitochondrial respiratory pathway is upregulated to produce more ATP. If the density of active Na/K pumps had been increased in response to the chronic Na+ influx in +/Lc Purkinje cells it seems reasonable to expect that the intensity of the histochemical labeling would have increased along Purkinje cell dendritic membranes. However, the staining pattern for Na/K pump activity remained diffuse in the +/Lc cerebellar cortex, with no evidence of discrete patches of labeling corresponding to the patchy labeling for α3 subunit observed in fluorescently labeled sections. The discrepancy between the distribution of α3 subunits detected by immunofluorescence and the pattern of Na/K pump histochemical activity could be due to limitations in the spatial resolution of the histochemical staining or some of the α3 immunolabeling labeling could represent deactivated Na/K pumps, including the 40 kD α3 cleaved isoform. In a mouse model of focal cerebral ischemia, no changes were found in protein or mRNA levels for Na/K pump isoforms [The diffuse pattern of Na/K pump activity in the +/ame ages . Individisoforms , but theα3 isoform into a 40 kD degradation product [α3 subunit and 40 kD degradation product increases in WT cerebella through P25 suggesting that a significant fraction of the native α3 subunit is oxidized and degraded by calpain in normal cerebellar tissue. Calpain has recently been shown to be active in individual, isolated +/Lc Purkinje cells at P14 based on immunolabeling for the 136 kD fragment of α-spectrin cleaved by calpain [Lc Purkinje cells at P15, there is no significant reduction in the levels of the 40 kD band in +/Lc cerebella until P25, when most Purkinje and granule cells have degenerated. As in the case of the 110 kD native α3 subunit, it is possible that the amount of the 40 kD degradation is increased in individual +/Lc Purkinje cells at P15 and P25, but total amounts are within control levels at P15 and significantly decreased at P25 because of the ongoing +/Lc Purkinje cell loss. +/Lc Purkinje cell degeneration is associated with an increase in oxidative stress, including an increase in mitochondrial cytochrome oxidase activity, which may generate more reactive oxygen species as a byproduct of increased respiratory activity. The results do not indicate that there is a dramatic increase in oxidized forms of the α3 subunit by P15 in the +/Lc cerebellum since there is not a significant increase in levels of the 40 kD byproduct, but the results are consistent with sustained proteolysis of the α3 subunit by calpain in +/Lc Purkinje cells until most have degenerated.There are a number of mechanisms that can reduce or irreversibly block Na/K pump activity. Na/K pump isoforms are routinely recycled by internalization in lysosomes and degradation by lysosomal cathepsin D , 38. How product . In this calpain . In thisLc Purkinje cells have shown that their degeneration is associated with increased expression of neuronal nitric oxide synthase and nitrotyrosine [α3 isoform, interactions with peroxynitrite, NO/PKG, and/or NO/cGMP reduce the density of active Na/K pumps. A reduction in the density of active Na/K pumps in +/Lc Purkinje cells may contribute to a Na+ overload in these neurons and exacerbate the cell death process, since increases in intracellular Na+ have been associated with induction of apoptosis [Na/K pump activity is also regulated by nitric oxide (NO)-cGMP  or NO-PKtyrosine . Since tpoptosis , 41.Lc Purkinje cells demonstrates that while the expression and/or distribution of the α3 subunit is disrupted in the cerebellar cortex of the +/Lc mutant, there is no evidence to suggest that the density of active pumps is increased in response to the constitutive influx of Na+ ions through the mutant GluRδ2Lc channels. We had hypothesized that Na/K pump activity would be stimulated as a homeostatic response to the Na+ ion influx. The lack of evidence for an increased density of functional Na/K pumps does not rule out the possibility that the activity of individual pumps is increased within each +/Lc Purkinje cell, thereby stimulating an increased demand for ATP which would eventually lead to depletion of cellular energy resources. The Na/K pump is estimated to use up to 50% of the ATP produced by neurons [+ is normally the rate-limiting factor in Na/K pump activity [+ within +/Lc Purkinje cells may increase the activity of individual pumps because of a more complete saturation of its Na+ binding sites. Thus, while there may be an increased ATP consumption in +/Lc Purkinje cells because of the increased activity of individual pumps, this study suggests that any increase in Na/K pump activity and ATP consumption is not due to an overall increase in the density of active ATPase pumps in chronically depolarized Purkinje cells.The analysis of the distribution and activity of the Na/K pump in this study of degenerating +/ neurons . Na+ is activity –45 and e


Nevertheless, this cell layer accounts only for about half of Fe stores in dry seeds. Comparison between Arabidopsis wild type (WT) and mutant seeds impaired in Fe vacuolar storage (vit1-1) or release (nramp3nramp4) confirmed the strongly altered Fe localization pattern in vit1-1, whereas no alteration could be detected in nramp3nramp4 dry seeds. Imaging of imbibed seeds indicates a dynamic localization of metals as Fe and Zn concentrations increase in the subepidermal cell layer of cotyledons after imbibition. The complementarities between μPIXE and other approaches as well as the importance of being able to quantify the patterns for the interpretation of mutant phenotypes are discussed.Seeds are a crucial stage in plant life. They contain the nutrients necessary to initiate the development of a new organism. Seeds also represent an important source of nutrient for human beings. Iron (Fe) and zinc (Zn) deficiencies affect over a billion people worldwide. It is therefore important to understand how these essential metals are stored in seeds. In this work, Particle-Induced X-ray Emission with the use of a focused ion beam (μPIXE) has been used to map and quantify essential metals in Arabidopsis seeds. In agreement with Synchrotron radiation X-ray fluorescence (SXRF) imaging and Perls/DAB staining, μPIXE maps confirmed the specific pattern of Fe and Mn localization in the endodermal and subepidermal cell layers in dry seeds, respectively. Moreover, μPIXE allows absolute quantification revealing that the Fe concentration in the endodermal cell layer reaches ~800 μg·g Thanks to their redox properties or Lewis acid strength under physiological conditions, iron (Fe), manganese (Mn), copper (Cu), or zinc (Zn) act as major cofactors in many enzymes such as proteases or antioxidant enzymes as well as in electron transfer chains of mitochondrial respiration or chloroplast photosynthesis . Importantly, μPIXE technique presents the unique advantage of providing quantitative results when used simultaneously with Rutherford Backscattering (RBS) and Scanning Transmission Ion Microscopy (STIM) analyses  allows multi-elemental mapping in biological samples with high spatial resolution (1 μm range) and high sensitivity  WT, nramp3nramp4 (nr3nr4) and vit1-1 mutants were used. Generation of both mutants has been described previously in Ravet et al. (−1), under continuous light (60 μmol photon·m−2·s−1) at 21°C for 48 h.Mature (dry) and imbibed seeds of Arabidopsis  at 80°C for 1 h, 100°C for 1 h, and 120°C for 2 h. After dilution to 12 ml with ultrapure water, K, Ca, Mn, Fe, Cu, and Zn contents of the samples were determined by atomic absorption spectrometry using an AA240FS flame spectrometer .In order to avoid losses of elements and their redistribution in the analysed samples, cryotechniques were used. The seeds were fixed by high pressure cryofixation  and stored in liquid nitrogen. Subsequent steps were performed in a cryostat  at −30°C: seeds were recovered from their cryofixation container in cold isopentane, embedded in an inert matrix  and cut with a tungsten blade in sections of approximately 30 μm. Sections were individually mounted on a cold aluminium sample holder covered with a pioloform film . Samples were carefully transferred to a freeze-dryer , freeze-dried at −10°C, under a vacuum of 0.37 mbar for 48 h and brought back to room temperature (RT) by steps of 5°C per hour. Finally, samples were covered by a second pioloform film and stored in an anhydrous environment at RT until μPIXE analyses. Section integrity of seed sample was verified by putting a few freshly cut sections directly on a microscope slide  stored in the cryostat. After sectioning, the microscope slides mounted with seed sections were quickly brought to RT in a vacuum desiccator and checked under a light microscope.−6 mbar), rough positioning of the samples was achieved using an optical camera. The proton beam of 3.0 MeV and 200 pA current was focused down to a 1.5 × 1.5 μm2 spot, used to rapidly scan large areas of the sample and finally adjusted to scan the seed section areas  during circa 4 h. Simultaneous PIXE, RBS, and STIM analyses were performed. A 40 mm2-Bruker XFlash SDD detector placed at 75° relative to the beam path and at 21 mm working distance of the scanned sample was used to collect X-rays emitted by non-organic elements. A 50 μm Mylar foil was placed in front of the detector to stop backscattered protons and attenuate X-ray signals from major elements. Backscattered particles were collected by a 180 mm2-surface barrier detector positioned at 170° and 35 mm from incident beam. Energy-loss maps based on STIM were obtained by collecting scattered particles at 30° using a surface barrier detector located behind the sample. In total, 11 seed sections were analyzed: 5 from WT, 5 from nramp3nramp4 mutant and 1 from vit1-1 mutant.Microanalyses of Arabidopsis sections were performed at the Saclay nuclear microprobe, France . For both dry and imbibed seeds, fractions of Fe and Mn that accumulate in their respective enriched areas compared to the whole seed section element content were calculated as followed: [element concentration in enriched area·area of enriched area]/[element concentration in whole seed section·area of whole seed section].Average elemental concentrations presented in Figures  as well Seed samples were prepared as recommended in Mesjasz-Przybylowicz and Przybylowicz  and descArabidopsis thaliana dry and imbibed seeds were analysed by Particle-Induced X-ray Emission induced by a focused ion beam (μPIXE) and elemental images were generated  to determine seed concentrations of various macro- and microelements  in the whole section, up to 751 μg·g−1DW in Fe-enriched area) and of Mn at the abaxial side of cotyledons (from 44 μg·g−1DW up to 386 μg·g−1DW). Surprisingly, slight accumulation of Zn also occurred in the Fe-enriched area (from 141 μg·g−1DW up to 311 μg·g−1DW). In vit1-1 dry seed, Fe (up to 360 μg·g−1DW) and Mn (up to 172 μg·g−1DW) concentrated in a single Fe- and Mn-enriched area at the abaxial side of the cotyledons, while no Fe accumulation around provascular tissues was observed. Here also, Zn (up to 224 μg·g−1DW) exhibited a slight accumulation within the combined enriched area.Elemental images obtained for the three genotypes showed that Fe distribution is strongly perturbed in e Figure . ROIs coe Figure . In WT anramp3nramp4 were measured. Statistical analyses of the results did not reveal any significant difference between WT and nramp3nramp4 in any of the ROIs analysed. Figure −1DW and 910 ± 74 μg·g−1DW) significantly accumulated in Fe-enriched provascular area as expected . Mn accumulated at significantly higher concentration in the Mn-enriched area at the abaxial side of the cotyledons of both dry and imbibed seeds  and Zn (268 ± 14 μg·g−1DW) were also significantly more concentrated within Mn-enriched area at the abaxial side of the cotyledons. These changes were not detectable visually on the elemental maps.The concentrations of Fe, Mn, and Zn in whole longitudinal and transversal seed sections as well as Fe- and Mn-enriched areas from dry and imbibed samples of WT and  Figures . As alreFinally, combining the relative areas of Fe- and Mn-enriched areas and the metal concentration within these areas, we calculated the fractions of Fe and Mn accumulated in their corresponding enriched areas, namely the provascular tissue region for Fe and the abaxial side of the cotyledons for Mn Table . These cnramp3nramp4 and vit1-1 mutant dry seeds. In addition, valuable information about local metal concentrations could be obtained from the analyses of the different types of spectra collected at the microprobe installation.Images of Arabidopsis dry seed sections obtained by μPIXE confirmed Fe, Mn, and Zn distribution patterns observed previously in WT, Fe distribution in Arabidopsis seeds has been already reported using different imaging techniques: EDX microanalysis on a transmission electron microscope (Lanquar et al., −1DW in provascular tissues were quantified by μPIXE (Table 3+ but this still remains to be demonstrated using XAS (Pich et al., Combined together, the information about Fe localization obtained by EDX, histochemical staining and SXRF techniques indicate that in Arabidopsis dry seed, Fe is concentrated in the endodermal cells of the embryo and, at subcellular level, is associated with vacuolar globoids (Lanquar et al., XE Table . As globXE Table , endoderImaging techniques also showed heterogeneous distribution of Mn and Zn in plant seeds (Kim et al., nramp3nramp4 mutant after seed imbibition was undertaken. Whereas EDX microanalyses revealed nramp3nramp4 mutant defect in Fe remobilization from globoids in 2-day old seedling, μPIXE failed to detect any qualitative or quantitative modification of the Fe pattern between the two genotypes at the same stage (Lanquar et al., During germination, seed undergoes drastic morphological and biological changes, necessarily inducing a redistribution of macro- and micronutrients Bewley, . WhereasNevertheless, quantification of Fe, Mn, and Zn in seed sections by μPIXE indicates significant distribution modifications after 48 h of imbibition. During imbibition, Fe and Zn concentrations increase in subepidermal cells of cotyledons, corresponding to the Mn-enriched area, while the fraction of Mn accumulating in those cells significantly increases from 30 to more than 50% of total Mn content. Although the origin of the metals could not be identified as no significant decrease of Fe, Mn, or Zn occurs in other areas, it is likely that during seed imbibition metals are already beginning to be redistributed toward the future photosynthetic cell layers. As the Fe concentration in the endodermal area is unchanged after imbibition, it is tempting to speculate that the Fe that accumulates in the sub epidermal area originates from the “diluted pool” spread in all seed tissues, which would therefore represent a more loosely bound Fe pool. The endodermal pool would be released at later stages of germination.In conclusion, our study using μPIXE complemented other approaches to analyse metal distribution in Arabidopsis seeds. This fully quantitative approach revealed unsuspected important Fe stores outside the endodermal cells. Analysis after seed imbibition provided a first glimpse at the rapid metal redistribution that occurs during germination.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.


Danaus plexippus) migrate up to 3000 km from their overwintering grounds in central Mexico to breed in eastern North America. Malcolm et al. (1993) articulated two non-mutually exclusive hypotheses to explain how Monarchs re-colonize North America each spring. The ‘successive brood’ hypothesis proposes that monarchs migrate from Mexico to the Gulf Coast, lay eggs and die, leaving northern re-colonization of the breeding range to subsequent generations. The ‘single sweep’ hypothesis proposes that overwintering monarchs continue to migrate northward after arriving on the Gulf coast and may reach the northern portion of the breeding range, laying eggs along the way. To examine these hypotheses, we sampled monarchs throughout the northern breeding range and combined stable-hydrogen isotopes (δD) to estimate natal origin with wing wear scores to differentiate between individuals born in the current vs. previous year. Similar to Malcolm et al. (1993), we found that the majority of the northern breeding range was re-colonized by the first generation of monarchs (90%). We also estimated that a small number of individuals (10%) originated directly from Mexico and, therefore adopted a sweep strategy. Contrary to Malcolm et al. (1993), we found that 62% of monarchs sampled in the Great Lakes originated from the Central U.S., suggesting that this region is important for sustaining production in the northern breeding areas. Our results provide new evidence of re-colonization patterns in monarchs and contribute important information towards identifying productive breeding regions of this unique migratory insect.Each year, millions of monarch butterflies ( Despite the massive scale of some insect migrations, remarkably little is known about how populations are spatially connected between different periods of the annual cycle (i.e. migratory connectivity) Danaus plexippus) migrate thousands of kilometres southward to discrete overwintering sites located between 2400–3600 m.a.s.l. in the Oyamel forests of central Mexico st spring generation. The ‘single sweep’ hypothesis (hereafter termed “SS strategy”) proposes that re-colonization is accomplished by the overwintering generation arriving at the Gulf Coast in early spring to lay eggs, but then continuing northward towards the Great Lakes, effectively recolonizing the entire eastern breeding range in a ‘single sweep’.Nearly the entire eastern North American population of monarch butterflies (Asclepias viridis) associated with the southern U.S.A., Malcolm et al. A. viridis is extensive and reaches as far north as Ohio Using cardenolide signatures of a milkweed species (st generation born in current year) strategy, we used stable-hydrogen isotopes (δD) to estimate natal origin Here, we examined these two re-colonization hypotheses by sampling monarchs from 44 sites across the Great Lakes region . To distn = 31; 14 sites), Michigan , Wisconsin , and Minnesota  and used by Malcolm et al Monarch adults were collected from May through June 2009 throughout the Great Lakes region: Ontario (2H) derived from high-temperature (1350°C) flash pyrolysis of wing tissue and continuous flow-isotope ratio mass spectrometry (CF-IRMS). Pure H2 was used as the sample analysis gas and the isotopic reference gas. A Hekatech HTO with an autosampler was used to automatically pyrolyse wing tissue samples to a single pulse of H2 gas (and N2 and CO gases). The resolved H2 sample pulse was then introduced to the isotope-ratio mass spectrometer ) via an open split capillary. Stable hydrogen isotopic ratios (2H/1H) of monarch wing tissue are reported in delta (δ) notation in parts per thousand (‰) deviation from the VSMOW-SLAP standard scale (Vienna Standard Mean Ocean Water-Standard Light Antarctic Precipitation) where δD = [(Ratiosample/Ratiostandard)−1×1000]. In order to determine the δD of non-exchangeable H2, we used the comparative equilibration method of Wassenaar and Hobson 2. Repeated within-run analyses of isotope laboratory standards yielded an analytical precision better than ±2.0‰ and ±0.3‰. The standards used for δD analysis and their within-analysis run precisions were CFS = −147.4±0.79‰ (n = 5), CHS = −187±0.56‰ (n = 5) and BWB = −108±0.33‰ (n = 5).Stable-hydrogen isotope (δD) analysis of wing chitin was conducted at the stable isotope laboratory at Environment Canada, Saskatoon, SK. Captured monarchs were euthanized and stored in paper envelopes. A small section of wing tissue was clipped from the hind wing of each monarch (350±10 µg) and encapsulated in 4.0×3.2 mm silver capsules. Stable hydrogen isotope measurements of samples and standards were performed on hydrogen (P) vary predictably along latitudinal gradients P isoscape.Stable-hydrogen isotopes are used as a marker for estimating the origin of animals in eastern North America because weighted mean growing-season δD values in precipitation  that feed on milkweed (R2 = 0.69) according to the linear function: δDm = 0.62δDp-79‰. We used the intercept of this function to adjust the δDP values for each breeding region to be equivalent δDm values In field-rearing experiments of monarchs, Hobson et al. y* = δDm and μ and σ are the mean and standard deviation of each breeding region calculated from the OIPC data as described above. Likelihood values for each monarch were normalized to generate a probability of assignment for a given breeding region relative to the sum of all other breeding regions:b* being the region with the highest probability. We also incorporated analytical error associated with δD measurements b* designations from the 100 re-samples , 12% had Pb* values (probability of originating from the assigned breeding region) >0.9 probability, 25% had 0.8 or higher, 52% had 0.7 or higher, and only 2% had Pb* values lower than 0.5. Summarizing the 100 re-samples for each monarch, 23% had Pb* assignments to the same region greater than 90% of time, 71% had >70% in the same region, and none had Pb* assignments to the same region less than 50% of the time  of monarchs that were not offspring born in the GL region followed the SB strategy and 10% (n = 11) followed the SS strategy. Surprisingly, of those that followed the SB strategy, 38% were from the GC region and 62% were from the CE region.Based on the assignment tests, we estimated that 36 monarchs originated from the Gulf Coast (GC) region and hence were considered to be 1tegy see . The remtegy see . Of the st generation individuals born in the Gulf Coast and Central regions of the U.S.A. (successive brood: SB) but that a small proportion of individuals (∼10%) originate directly from Mexican over wintering sites (single sweep: SS). Our estimate of the proportion of SB individuals (90%) is in remarkable agreement with that reported by Malcolm et al. We provide evidence that monarchs adopt one of two distinct migratory strategies during the northward migration unpublished data), we estimated that monarchs born in this region would be no older than 24 days . This is likely insufficient time for monarchs to develop a high degree of wear, particularly if we consider that individuals returning from over wintering sites that were sampled on the Gulf Coast had wing wear scores of 3.28 (±0.83) and monarchs that were sampled in the Great Lakes region that were born in the Gulf Coast the same year had wing wear scores of 2.94 (±1.16) Our assignment of migration strategy was based on a combination of stable isotope values and wing wear. Although the confidence in assignments to specific breeding regions based on stable isotope values was high, it is possible that some monarchs were mis-assigned to have been born in the previous year or sampling year due to some uncertainty in the relationship between wing wear and age. Of the monarchs estimated to have originated from the Gulf Coast region, 75% (n = 27/36) had wing wear scores within the expected range (1–3). However, it is unlikely that the 25% (n = 9) of Gulf Coast monarchs with wing wear ≥4 were mis-assigned since milkweed does not grow in the Gulf Coast during late summer so they could not have been born there the previous year. The most likely opportunity for a year mis-assignment existed with monarchs originating from the Central region. However, the phenology of monarch arrival in this region and wing wear data suggests that the probability of error is likely low. The earliest observation of monarch eggs within the Central region during the spring of 2009 was on April 4 at the extreme southern edge of this area unpublished data) and found that SS monarchs may account for as much as 25% of monarchs arriving in the Great Lakes region during spring. Hence, SS monarchs could contribute a significant proportion of offspring to this area during June and early July but more thorough sampling throughout the entire spring period would help resolve this uncertainty.It is possible that we underestimated the proportion of monarchs adopting a single sweep strategy because our sampling took place only once at each site during the spring. Hence, monarchs originating from over wintering sites could have died soon after arrival but prior to our sampling. We ran a simulation model to examine this possibility  and 82% of all over wintering SS monarchs born the previous year (9/11) were born in the CE region. This also suggested that SS monarchs were migrating further than 35°N to produce a large cohort of SB monarchs in the Central region and that this area is likely an important breeding ground for fall migrants travelling to Mexico in late summer. This area of high monarch productivity corresponds with the results of Wassenaar and Hobson In contrast to previous work that suggests that the majority of monarchs arriving in the Great Lakes region in spring originate from south of 35°N , we found that 64% of individuals (68/106) were born between 35°N–41°N in the Central part of the eastern U.S.A. . Of these, 62% of 1Previous work has suggested that conservation efforts aimed at protecting habitat in the Gulf Coast may be particularly important for monarchs following a SB migration strategy since these areas are thought to be the primary breeding grounds of this life-history strategy Despite the long-standing mysteries surrounding the spring migration of the monarch, even less is known about the migrations of many other insect species of dragonflies, moths, butterflies, and aphids that also migrate north during the spring. Understanding such migrations is even more important when considering that many of the major insect pests which cause billions of dollars of damage to the agricultural industry each year are also migratory Our research helps to resolve the degree to which individuals utilize different migration strategies to re-colonize eastern North America each spring. In doing so, we provide critical information needed for conservation planning of this species by identifying important breeding locations for the monarch during the northward migration from Mexico.


We also compared the δ13C and δ15N values among four populations of S. guianensis. Variation in carbon isotope results from coast to ocean indicated that there was a significant decrease in δ13C values from estuarine dolphins to oceanic species. S. guianensis from Guanabara Bay had the highest mean δ13C value, while oceanic species showed significantly lower δ13C values. The highest δ15N values were observed for P. crassidens and T. truncatus, suggesting that these species occupy the highest trophic position among the delphinids studied here. The oceanic species S. attenuata, G. griseus and L. hosei had the lowest δ15N values. Stable isotope analysis showed that the three populations of S. guianensis in coastal bays had different δ13C values, but similar δ15N results. Guiana dolphins from Sepetiba and Ilha Grande bays had different foraging habitat, with specimens from Ilha Grande showing more negative δ13C values. This study provides further information on the feeding ecology of delphinids occurring in southeastern Brazil, with evidence of distinctive foraging habitats and the occupation of different ecological niches by these species in the study area.To investigate the foraging habitats of delphinids in southeastern Brazil, we analyzed stable carbon (δ Sotalia guianensis), as well as along the continental shelf and in oceanic environments off the coast of Rio de Janeiro State. However, there is little information regarding the habitat preferences and feeding ecology of delphinids from the study area. Most of the species, including false killer whale (Pseudorca crassidens), Risso's dolphin (Grampus griseus), spinner dolphin (Stenella longirostris), Fraser's dolphin (Lagenodelphis hosei) and pantropical spotted dolphin (Stenella attenuata), have been observed opportunistically because they usually occupy off-shore areas. Investigating the habitat preferences and the trophic relationships among the delphinid species is of great importance for understanding the roles and ecological niches occupied by these animals in marine food webs. This information will make it possible to better understand the degree of overlap and segregation of delphinids in the foraging area in southeastern Brazil.Delphinidae constitutes the richest taxonomical family of all cetaceans, with 36 currently recognized species. The presence of delphinids along the Rio de Janeiro coast has been reported from direct observation or from stranding records Rio de Janeiro State is located along the southeastern Brazilian coast. This region is under high anthropogenic pressure because it is an important urban and industrial center for Brazil The more traditional methods used for studying the feeding ecology of cetaceans relies on stomach content analyses from stranded or accidentally caught animals 13C) values have been used to trace the primary source of carbon in the food web because this isotope is indicative of low trophic enrichment (1–2‰) 13C values of particulate organic matter (POM) and phytoplankton can vary along a gradient of coastal to oceanic regions, with higher δ13C values in waters closer to the coast 15N) have been used to study trophic relationships in marine food webs and to assess trophic levels 15N values and the trophic position that an organism occupies The analysis of carbon and nitrogen stable isotopes has been shown to be a useful complementary tool for investigating foraging and feeding behavior of cetaceans Stable carbon and nitrogen isotope analyses were performed using delphinid muscle to 1) investigate the foraging area and trophic relationships of 10 delphinid species from southeastern Brazil, 2) compare the stable isotope values among four Guiana dolphin populations from the coast of Rio de Janeiro State, and 3) identify possible trophic differences between sexes and among age classes of Guiana dolphins.Muscle samples of delphinids were collected with appropriate permissions from Brazilian Environmental Agencies – IBAMA/MMA (permission number 11495-1) and ICMBio/MMA (permission number 11579-1).Stenella frontalis (n = 13), bottlenose dolphin, Tursiops truncatus (n = 7), rough-toothed dolphin, Steno bredanensis (n = 3), false killer whale, Pseudorca crassidens (n = 2), common dolphin, Delphinus sp. (n = 2), Fraser's dolphin, Lagenodelphis hosei (n = 10), pantropical spotted dolphin, Stenella attenuata (n = 2), spinner dolphin, Stenella longirostris (n = 1), and Risso's dolphin, Grampus griseus (n = 1), and 4 populations of Guiana dolphin, Sotalia guianensis, from Guanabara Bay (n = 26), Sepetiba Bay (n = 49), Ilha Grande Bay (n = 10) and “Região dos Lagos” (n = 5).Muscle samples of 10 delphinids species  were obtained from specimens either incidentally caught in gillnet fisheries or stranded on the beaches of Rio de Janeiro State in southeastern Brazil from 1994 to 2009 . The fol13C or 15N and R is the corresponding ratio of 13C/12C or 15N/14N. Carbon and nitrogen ratios were expressed in relationship to the V-PDB (Vienna Peedee Belemnite) standard and to atmospheric nitrogen, respectively. Reference materials (IAEA CH-6 and IAEA-N1) were also analyzed. The standard deviation on replicated measurements from a single delphinid sample was ±0.3‰.Muscle samples were dried at 60°C for 72 h and then ground into a homogeneous powder. Dried samples (∼1.5 mg) were weighed and placed in tin capsules (3×5 mm), and carbon and nitrogen stable isotope measurements were performed on a V.G. Optima (Isoprime UK) isotope ratio mass spectrometer coupled to an N-C-S elemental analyzer (Carlo Erba). Stable isotope ratios were expressed in delta notation as parts per thousand according to the following equation: 13C and lipid tissue content can be variable 13C values according to the following equation Because lipids have been shown to be depleted in post-hoc test, were used to compare carbon and nitrogen isotope values among species; dolphin calves were excluded from these analyses. In addition, we performed a cluster analysis aiming to detect isotopic patterns among delphinids species. For this analysis, we used Ward's method (minimum variance) and Euclidean distances 13C and δ15N values among adult males, adult females and calves of Guiana dolphins from Guanabara and Sepetiba bays. The Student's t-test was performed to compare male and female dolphins from Ilha Grande Bay.The Kolmogorov-Smirnov test was used to test for normality of the data . Analyses of variance (ANOVA), followed by an Unequal N HSD 13C and δ15N values from the 10 delphinids species from the Rio de Janeiro State coast ranged from −17.1 to −13.8‰ and from 11.3 to 15.3‰, respectively  = 18.64, p<0.0001 and δ15N: F = 7.04, p<0.0001)  and the fourth occurs along the coast in an area known as “Região dos Lagos”. Mean δectively , Fig. 2.<0.0001) . Statist13C values, while dolphins from Sepetiba Bay and “Região dos Lagos” had intermediate values and individuals from the Ilha Grande Bay had the lowest δ13C values    . However p>0.99) .13C values than Atlantic spotted, bottlenose and Fraser's dolphins . We found significant differences between the oceanic species  and Guiana dolphins for both δ13C and δ15N values  identified five groups among the delphinid species  = 0.26; p = 0.77), although there was a difference in δ15N values (ANOVA F(2.22) = 6.44; p = 0.006). Calves showed higher δ15N values in relation to adult males and females; the adult males and females themselves did not show differences  (13C (ANOVA F(2.44) = 4.93; p = 0.011) and δ15N values (ANOVA F(2.44) 34.99; p<0.00001) . Calves had higher δ15N values than adults . For specimens from Ilha Grande Bay, the only possible comparison performed was between adult males and females due to the absence of calf samples. Values of δ13C and δ15N were similar for both sexes .We compared adult males, adult females and calves from Guanabara and Sepetiba bays. There was no significant difference in δctively) . There w0.00001) . The pos13C values revealed differences between species and allowed us to define groups according to their foraging habitat. There was a significant decrease in δ13C values from estuarine dolphins to oceanic species, indicating coast-ocean variation in isotopic ratios. Similar results were observed in other studies involving cetaceans 13C values in primary sources of carbon in food webs, with coastal and/or benthic systems having higher values than oceanic and/or pelagic systems Analysis of carbon isotopes has proven to be a very useful tool for identifying differences in both inter-13C values, even when compared with the same species from Sepetiba and Ilha Grande bays. For dolphins from Guanabara Bay, high site fidelity 13C values.Guiana dolphin is a species that inhabits estuarine and coastal regions throughout its distribution 13C-depleted values. δ13C values point to two distinct ecological populations in Sepetiba and Ilha Grande bay. These findings corroborate results from previous studies showing differences in the accumulation of organochlorine compounds Site fidelity of Guiana dolphin has also been observed in Sepetiba Bay 13C values of Guiana dolphins from Sepetiba Bay varied widely, ranging from −16.9‰ to −12.8‰. This finding may indicate the existence of distinct food sources for this population, suggesting that some individuals forage outside the bay rather than feeding exclusively within Sepetiba Bay. Dias et al. The δ13C values compared to specimens from the other bays investigated, with values close to those of oceanic delphinids. It is important to highlight that the species is typically a coastal species and, to date, no sighting has been described in the oceanic environment 13C-depleted. Furthermore, Ilha Grande Bay is a semi-open system that is more heavily influenced by the colder, more saline water from the marine current flowing from the continental shelf than are Guanabara and Sepetiba bays 13C values in Guiana dolphins from Ilha Grande Bay were due to the influence of external water in this estuarine environment.Guiana dolphins from Ilha Grande Bay had lower δ15N values, indicating that they are feeding on prey from the same trophic level. However, it is believed that there may be differences in the trophic position of these populations due to variation in the nitrogen isotopic composition at the base of the food webs among different systems. Bisi et al. 15N values were reduced at the base of the Guanabara Bay food web. The same authors verified that Guiana dolphins from Guanabara Bay occupy the top trophic level of the food web in this estuary, exhibiting the highest δ15N values among the different organisms studied. In contrast, the specimens from Sepetiba Bay are feeding on organisms that occupy relatively lower trophic levels 15N values to those of other populations of the species, they may occupy a higher trophic position in the food web.The four populations of Guiana dolphins had similar δ15N values than adults in Guanabara and Sepetiba bays. These findings are probably due to isotopic fractionation during the assimilation of breast milk, as calves occupy a higher “trophic level” than their mothers during nursing periods Previous studies on marine mammals have shown that feeding ecology may or may not vary between males and females 13C values of organic carbon dissolved in waters under the influence of SACW have been observed 13C values between Guiana dolphins from Ilha Grande Bay and Atlantic spotted dolphins suggest that SACW influences the foraging areas of these two species along the coast of Rio de Janeiro State.Guiana dolphins from Ilha Grande Bay and the Atlantic spotted dolphin were grouped by cluster analysis. Except for two specimens, the Atlantic spotted dolphins sampled were obtained from beaches of “Região dos Lagos”, an area influenced by the South Atlantic Central Water (SACW) upwelling during the summer 13C analysis suggest that the species uses continental shelf waters in southeastern Brazil, primarily foraging along the inner part of the shelf. This hypothesis is reinforced by the results of the cluster analysis, in which Guiana and rough-toothed dolphins shared the same group.Rough-toothed dolphins are typically found in oceanic regions 13C and δ15N values. These species had the highest δ15N values, suggesting that these animals occupy the highest trophic level among the delphinids considered in this study. Stomach content analyses showed that bottlenose dolphins feed mainly on teleost fish and cephalopods along the south-central coast of Rio de Janeiro State 15N values 15N values are probably feeding specifically on cephalopods, whereas individuals that are 15N-enriched would be feeding at higher trophic levels  15N values found in this study, it is likely that false killer whales prey mainly on high-trophic-level fish or even on marine mammals. These results are in accordance with the high concentrations of organohalogen compounds found in tissues of false killer whales from the study region, which suggest regular feeding on marine mammals 13C values to other nearshore species (rough-toothed and Guiana dolphins), suggesting that these species also forage in the region along the continental shelf, with similar habitat preferences. This is a relevant finding because studies have reported the use of oceanic habitats, in waters of greater than 1,000 m, for false killer whales Bottlenose dolphins and false killer whales occupied similar trophic niches, with similarities in δ13C values indicated that Risso's and pantropical spotted dolphins inhabit waters along the continental shelf break. A similar result was observed for Risso's dolphins in Tierra del Fuego, Argentina 13C values, lending further support to the described use of oceanic habit 15N values and were found in groups four  and five (spinner dolphin and Fraser's dolphin), identified from the cluster analysis. Studies have shown a positive correlation between trophic level and δ15N values 15N values of the isotopic baseline can vary considerably among ecosystems and regions 15N results may reflect oceanic species feeding on low trophic level prey or could be due to the low δ15N values at the base of the ocean food web. An important source of nitrogen in the ocean's photic zone is in the form of nitrate, which typically features higher δ15N values of approximately 6 ‰ 15N values in the biota to the influence of atmospheric N2 fixation by cyanobacteria in oceanic waters 15N values in oceanic dolphin species point to a substantial input of N2 fixed by cyanobacteria rather than nitrate as a primary source of nitrogen in foraging areas.The δ15N values. Stomach content analyses have shown that pantropical spotted dolphins feed mainly on mesopelagic fish of the Myctophidae family, as well as on cephalopods from the families Enoploteuthidae and Ommastrephidae 13C and δ15N values were very similar among pantropical spotted and Risso's dolphins, suggesting a large overlap in foraging area or prey consumed.Among the oceanic species, pantropical spotted and Risso's dolphins had the lowest δ13C and δ15N values indicated that there was segregation among the delphinids occurring along the coast of Rio de Janeiro State, with species having distinctive foraging habitats and occupying different ecological niches. For example, rough-toothed dolphins appear to forage along the inner shelf, whereas bottlenose dolphins and false killer whales use the continental shelf. Values of δ13C suggest that Risso's and pantropical spotted dolphins forage along the platform break, while spinner and Fraser's dolphins used similar oceanic habitat. Bottlenose dolphins and false killer whales occupied the highest trophic position, while spinner and Fraser's dolphins fed on lower trophic level prey. However, investigations regarding the δ15N values at the base of food webs in different environments are necessary for a better understanding of the trophic levels occupied by delphinid species. Lastly, δ13C values showed a clear separation between the Guiana dolphin populations from adjacent areas. It is important to emphasize that the delphinid species studied occur in a region under high anthropogenic pressure, subject to pollution, intense vessel traffic, oil exploration, seismic prospecting, and intense commercial fishing, among other factors. Knowledge and understanding of the habitat preferences of delphinids in southeastern Brazil is of fundamental importance for identifying potential threats to which these animals are subjected, as well as for supporting appropriate conservation actions.This study provides new information on the trophic ecology of 10 delphinid species, including four populations of Guiana dolphins, in southeastern Brazil. Evidence from δ


T. thermophilus 30S ribosomal subunit crystals is reported.Serial femtosecond X-ray (SFX) diffraction extending beyond 6 Å resolution using  High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes. The early challenges in obtaining crystals diffracting to a resolution sufficient to provide useful information were overcome at the end of the 1990s  using X-ray free-electron lasers (XFELs)  software suite.30S ribosomal subunits from 2.2.−1 was injected into the interaction region inside a vacuum chamber at the CXI instrument  MPD. After harvesting in the same mother liquor, microcrystals of 3 × 5 × 200 µm in size were pooled and suspensions were prefiltered through a 20 µm Upchurch stainless-steel filter to remove large particles and aggregates . This filtration process was followed by a second filtration through a Millipore Isopore polycarbonate screen filter to optimize crystal size distribution, limiting the crystal width to approximately 1–5 µm and the crystal length to approximately 3–20 µm . A second 20 µm Upchurch filter used for pre-filtration was used as an in-line filter during data collection.For the SFX experiments, the hanging-drop crystallization conditions were optimized to favor the formation of microcrystals by increasing the precipitant concentration in the crystallization buffer from 14% to 17%. This resolution is less than that obtained using synchrotron X-rays under cryogenic conditions. This may be accounted for, in part, by the harsh treatment of the crystals in the particular experimental setup available at the time of the experiment. The crystals had been subjected to high pressure and large mechanical shearing forces during repeated filtrations to reduce the potential for clogging of the GDVN microjet during injection across the XFEL beam. These repeated physical contacts with filters and high pressure, at ∼10.3 MPa from the HPLC-pump driven GDVN, can damage the packing arrangement of the 30S ribosomal subunits in the crystal lattice, introduce additional mosaicity to the crystals and thereby lower the resolution limit. Future experiments will employ a gentler and much lower pressure injection system such as an electrospinning microjet with 50–150 µm inner capillary options , consistent with the known parameters and space group (P41212) for these crystals obtained by conventional cryocrystallography (Wimberly et al., 2000Despite the low resolution of the recorded diffraction, it was possible to confirm the unit-cell parameters using the


Leptin is required for optimal health in a number of physiological systems . Despite the extensive leptin literature and many observations of leptin’s cyclical pattern over the 24-hour day, few studies have specifically examined how the circadian rhythm of leptin may be essential to leptin signaling and health. Here we present data indicating that a rhythmic leptin profile (e.g. 1 peak every 24 hours) leads to excessive weight gain during desynchronized feeding whereas non-rhythmic leptin provided in a continuous manner does not lead to excessive body weight gain under similar feeding conditions. This study suggests that feeding time can interact with leptin’s endogenous rhythm to influence metabolic signals, specifically leading to excessive body weight gains during ‘wrongly’ timed feeding.The neuroendocrine and metabolic effects of leptin have been extensively researched since the discovery, and the later identification, of the leptin gene mutated within the  The master circadian clock, located within the surprachiasmtic nuclei (SCN), orchestrates the cyclic rhythms of many behaviors, tissues, hormones, genes, and other physiological processes. Studies over the last decade have demonstrated that the molecular clock mechanism operates in a cell autonomous fashion in most, if not all, peripheral tissues In both humans and rodents, feeding during the ‘wrong’ circadian time has been implicated in body weight gain. Night-shift workers, who must be awake, active, and eating during the night are at a higher risk for obesity and cardiometabolic diseases With nocturnal rodents, a desynchronized feeding (DF) protocol A wild type B6 mouse challenged with DF will gain significantly more weight than a B6 mouse fed only during the normal circadian feeding phase Since its discovery, leptin has been indicated as an important component of metabolism, reproduction, motivated behaviors, and others in vitro suggesting a bidirectional relationship between the circadian clock and leptin Naturally occurring fluctuations in leptin levels occur over the period of a day and are closely associated with feeding and insulin release in both humans and rodents ob/ob mouse, on a C57BL/6J (B6) genetic background, which has a mutation in the leptin gene rendering it ineffective throughout the mouse. As a result of the absence of leptin, ob/ob mice are hyperphagic, obese, and develop characteristics associated with the metabolic syndrome in humans including elevated glucose, and insulin insensitivity ob/ob mouse to test the hypothesis that non-rhythmic continuous leptin will have differential effects on body weight gain compared to rhythmic leptin. We present data indicating that the presence of leptin alone is not sufficient to lead to weight gain from DF, instead, the presence of leptin and a rhythmic peaking of leptin is necessary for weight gain to occur from DF suggesting that the endogenous leptin rhythm is a possible mechanism leading to excessive weight gains from ‘wrongly’ timed feeding.Despite a wealth of literature describing the natural circadian variation in leptin over the 24-hour day, surprisingly few studies have examined the functional significance of the circadian leptin rhythm for health and treatment (e.g. chronotherapy) ob/ob mice do not gain excess weight during DF . Interestingly, continuous leptin failed to cause excessive weight gain during DF  in mice (e.g. light-phase feeding). As our previous study demonstrated uring DF . Howeverob/ob mice were placed on DF and monitored for body weight, food intake, and activity levels. To control for the length of feeding and fasting periods, DF ob/ob mice are compared to synchronized feeding (SF) ob/ob mice that have access to food for the same length of time but only during the normal circadian feeding period (e.g. the dark phase).To test the hypothesis that leptin may be central to the weight gain seen during ‘wrongly’ timed feeding, leptin-deficient ob/ob were similar under both DF and SF conditions. The timing of the high-fat diet failed to affect body weight gains in leptin deficient ob/ob mice  = 1306.1, p<0.001) indicating that all ob/ob mice gain weight while on the high-fat diet.As expected, both DF and SF ob/ob mice  = 0.49, p = 0.50). Similarly, no differences were seen in cumulative caloric intake between DF  and SF  ob/ob mice over the six week period  = −0.67, p = 0.52). . t(14) = 0.51, p = 0.62). However, there was significant difference in the amount of activity occurring during the circadian dark phase: the DF group had significantly less activity during the dark phase than the SF group  = −2.50, p = 0.03). This decrease in dark activity was not accompanied by a statistically significant increase in light phase activity within the DF group most likely due to the large within group variation  = 0.85, p = 0.41).Over the course of the six week experiment, no differences were seen in cumulative activity counts  for two weeks. This length of time was chosen based on previous data indicating that B6 mice on DF gain significantly more weight than SF mice within two weeks To test the hypothesis that the presence of leptin alone could lead to excessive weight gains from DF in the ob/ob mice weighed the same as SF ob/ob mice suggesting that the presence of leptin alone was not sufficient to lead to weight gain from DF as seen in wild type mice. The timing of feeding did not affect body weight in ob/ob mice provided non-rhythmic leptin  = 0.42, p = 0.53). Similarly, no differences were seen in the cumulative caloric intake between DF  and SF  ob/ob non-rhythmic leptin treated mice over the two week period  = −0.65, p = 0.53). . t(12) = -0.64, p = 0.53). Similarly, no statistical differences were observed in the amount of activity occurring during the circadian light phase  = 1.23, p = 0.24) or the circadian dark phase  = −1.40, p = 0.19). However, as in the previous experiment, the SF group appears to favor being active during the circadian dark phase more than the DF group.Over the course of the two week experiment, no differences were seen in cumulative activity counts . In addition to examining the effects of feeding time (e.g. the DF and SF groups), we also examined the timing of the leptin injection. Half of each feeding group were given a leptin injection during the feeding phase to produce a naturally occurring feeding-associated leptin peak (synchronized leptin  =  SL) while the other half of the feeding group were given a leptin injection during the fasting period to produce an abnormally occurring leptin peak desynchronized from feeding (desynchronized leptin  =  DL). Therefore, the four experimental groups (each N  =  8) were as follows:To test the hypothesis that rhythmic leptin is necessary to lead to excessive weight gain from DF, we gave a once daily leptin injection to SF-SL, having normally timed feeding and a normal feeding-associated leptin peak,SF-DL, having normally timed feeding and a feeding-dissociated leptin peak,DF-SL, having ‘wrongly’ timed feeding and a normal feeding-associated leptin peak, andDF-DL, having ‘wrongly’ timed feeding and a feeding-dissociated leptin peak.ob/ob mice gain significantly more weight than SF ob/ob mice (ob/ob mice within the most circadian desynchronized DF-DL group gained the most weight and were significantly different from the most synchronized SF-SL group (p = 0.01) and the moderately synchronized SF-DL group (p = 0.04). Additionally, DF-SL mice gained more than SF-SL mice (p<0.05). When focusing on the weight gains at the end of the two week study  = 3.55, p = 0.03), DF-DL gained significantly more than SF-SL (p = 0.01) and SF-DL mice (p = 0.01). Additionally, DF-SL mice showed a trend to gain more than SF-DL (p  =  0.08) and SF-SL mice (p = 0.07), suggesting that a desynchronized feeding rhythm leads to more metabolic impairments than a desynchronized leptin rhythm.Only when leptin was given by daily injection did DF ob/ob mice  = 0.71, p = 0.41) suggesting that a desynchronized leptin rhythm by itself would not lead to significant weight change without the additional component of ‘wrongly’ timed feeding. However, the timing of the leptin peak may influence body weight to some degree as represented by the beginning of a separation of body weight gains between the DF-DL and DF-SL groups.Interestingly, leptin injection time by feeding-association (DL vs. SL) alone had no effect on body weight gains in ob/ob mice gain weight differently over the two week period than SF ob/ob mice regardless of leptin injection time.As expected, all groups gained weight over the duration of the study  = 601.42, p<0.001). There was a significant interaction effect  = 5.4722, p<0.001) between feeding phase and study duration suggesting that the DF Of the four groups, no differences were seen in overall weekly caloric intake  = 1.50, p = 0.24). Similarly, an one-way ANOVA of the cumulative caloric intake over the two week period failed to show any differences in caloric intake  = 1.50, p = 0.24) among the four groups . Neither feeding time  = 1.78, p = 0.19), leptin injection time  = 0.13, p = 0.72), or their interaction  = 2.60, p = 0.12) had an effect on caloric intake. Over the course of the experiment, no differences were seen in cumulative activity counts  in the present study, we also integrated differences in feeding time with the rhythmic or non-rhythmic behavior of the metabolic hormone leptin. Notably, we examine two circadian aspects of leptin expression: 1) rhythmic vs. non-rhythmic expression and 2) synchrony vs. desynchrony with feeding behavior. Within normal, healthy humans and rodents, leptin is naturally rhythmic and associated with feeding time . In obese individuals, leptin levels are elevated but the rhythm of leptin remains relatively unchanged with the exception of a reduction in amplitude ob/ob mice . When rhythmic leptin was present, ob/ob mice gained excessive weight during DF. As the endogenous leptin profile of wild type mice is also rhythmic, it is possible that the circadian expression of leptin provides additional cues leading to excessive body weights from DF. The data from experiment 3 indicate that a rhythmic profile of leptin is needed to induce weight gains from DF. As an alternative explanation, these data also suggest that rhythmic leptin administration is less effective at preventing weight gain than continuous leptin administration. Indeed, ob/ob provided continuous leptin gain less weight over time than ob/ob mice given the same amount of leptin in a single daily dose.Given that non-rhythmic leptin failed to increase weight gain during DF, we next examined if the presence of a leptin rhythm could lead to increased weight gains during DF. To create a leptin rhythm in the Ob/ob mice fed in the dark and receiving a leptin injection during the dark (SF-SL) would closely mirror a normal wild type mouse which has its feeding behavior synchronized to the light:dark cycle and its leptin rhythm synchronized to the feeding behavior. At the opposite extreme is the DF-DL group that was fed exclusively during the ‘wrong’ phase (e.g. light phase) and received leptin injections dissociated from the feeding time (e.g. during the dark phase). Intriguingly, these two groups showed the most profound differences in body weight with the DF-DL mice gaining the most weight and the SF-SL mice gaining the least amount of weight over time. These data suggest that minimal circadian disorganization can lead to optimal body weight regulation whereas maximal circadian disorganization can lead to the greatest impairments in body weight regulation . However, we observe increased body weight (p<0.05) in mice eating during the light phase (and receiving leptin at that time) than in mice eating during the dark phase (and receiving leptin at that time).Statistical analyses of the two variables “feeding synchrony” and “leptin synchrony,” indicate that only feeding synchrony will lead to differential weight gain during DF. This suggests that circadian mistimed feeding will lead to a greater impairment of body weight regulation than a mistimed leptin peak. This in conjunction with experiments 1 and 2 suggests that while a rhythm of leptin is necessary to induce increased weight gains from ‘wrongly’ timed feeding, the actual time of the leptin peak is not essential. This is intriguing and mirrors other rhythmic hormones such as GnRH, which is known to have little signaling effect when given continuously but signals robustly when given rhythmically, regardless of the circadian time of day that rhythm is given It is interesting to note that the mice receiving the once daily leptin injection consumed less calories but gained more weight than the mice receiving the constant release leptin pump. This relative decrease in caloric intake may be due to the presence of the injection  during the feeding phase which leads to increased arousal, exploratory activity, and other stereotypical behaviors including an acute affect on feeding. The relative increase in body weight within the leptin injection mice suggests a different utilization of calories within the leptin injection mice, one that intensely favors weight gain and possibly fat storage.ob/ob mice having no leptin or receiving non-rhythmic leptin do not gain increased body weight from ‘wrongly’ timed feeding. Within a wild type mouse, for example, fluctuations in leptin and leptin receptor number and expression naturally occur coincident with feeding, however when leptin fluctuations occur during ‘wrongly’ timed feeding increased weight gain follows, perhaps through interactions with food-driven mechanisms and/or other circadian-driven mechanisms. Indeed, other research has hypothesized that a major role the leptin circadian rhythm may be to maintain a set threshold of leptin to order to prevent excessive caloric intake The rising and falling of leptin levels over the 24-hour day trigger fluctuations in other feeding hormones such as ghrelin Our results also indicate that despite both DF and SF mice receiving the same length of fasting (12 hours), fasting during the dark phase (and feeding during the light) leads to a greater drop in leptin than fasting during the light . However, it is possible that confounding metabolic factors present in the ob/ob mouse may lead to misinterpretation of the data. Therefore, additional experiments using wild type mice or another non-metabolic model would make ideal follow-up studies.It is important to highlight that the To our knowledge, these data present the first evidence that leptin has differential effects depending on a peak in leptin levels. One previous study did examine the treatment effects of a leptin injection given in the morning, evening, or both in the morning and evening in obese humans It is interesting to integrate our results from an evolutionary prospective. It would be maladaptive, for instance, for a nocturnal animal to be awake and seeking food during the daytime when predators are readily available. But if a nocturnal animal critically requires calories, it may override the circadian clock (therefore being awake and eating during the ‘wrong’ circadian time) and it would then be adaptive to utilize the forged calories in such a way so to limit future food seeking behavior during the ‘wrong’ time. This can be accomplished by storing the calories and increasing body weight. An increase in body weight is precisely what happens during desynchronized feeding and it could be the endogenous leptin rhythm coupled to the feeding time that promotes such a response. Perhaps once a beneficial response promoting fitness, body weight gain from ‘wrongly’ timed feeding now has detrimental implications for many humans consuming a significant portion of their calories during non-ideal circadian times, including non-breakfast eaters All experimental mice were housed and handled according to the Federal Animal Welfare guidelines. All studies were conducted at Northwestern University using procedures approved in advance by the Institutional Animal Care and Use Committee, university assurance number A3283-01.ob/ob mice were ordered from Jackson Laboratory  at 4–5 weeks of age and immediately singly-housed within temperature, light, and humidity controlled light boxes on a 12 hour light, 12 hour dark schedule. Mice begin experimentation at 6-weeks of age after having one week of environmental acclimation. On average, ob/ob mice were ∼30g when beginning the experiment. Male C57BL/6J (B6) mice were bred at Northwestern University and began experimentation at 10–11 weeks old within temperature, light, and humidity controlled light boxes on a 12 hour light, 12 hour dark schedule. On average, B6 mice were ∼24.5 g when beginning the experiment.Male ob/ob mice were allowed continuous free-access to food and were maintained on the standard chow diet in order to limit weight gain during ad libitum feeding.All mice were initially fed a standard chow  of which 16% of the calories are from fat until the start of the desynchronized feeding protocol. After the start of desynchronized feeding, mice were fed a high-fat diet in which 60% of the calories are from fat . A subset of Desynchronized Feeding. Mice were placed onto a restricted feeding protocol as previously described 14-day constant release leptin pumps  were filled with a 100ug/kg/day leptin solution containing 0.9% saline solution. Pumps were activated in a warm saline bath for 12 hours prior to implantation. Pumps were implanted subcutaneously into isofluane-anesthetized mice in the dorsal interscapular region just prior to their designated feeding phase. Previous studies have indicated that an osmotic pump delivers leptin stably and consistently ob/ob mice to wild type levels during the same 4-6 hr period similarly fed wild type would experience a physiological leptin peak. A saline injection was given at the counter ZT hour to control for the stress of injection at both ZT4 and ZT16. It was previously determined that the timing of the leptin injection alone while on ad libitum standard chow feeding did not lead to differences in body weight  or ZT16 (i.e. four hours after dark onset) at the same dose as the leptin pumps, 100 ug/kg/day. Both the dose and the timing of the leptin injections were chosen to mirror the physiological leptin levels of wild type mice fed the same SF or DF protocol. The single 100 ug/kg/day leptin dose was determined sufficient to slow weight gain while on a high-fat diet , to be ay weight .Activity. Overall activity was recorded continuously using a one-dimensional, three-beam infrared (IR) monitoring board. Beam breaks were recorded and data analyzed using an online recording program .Unless otherwise stated, data were analyzed using a repeated measured ANOVA with the aid of a statistical program .Ob/ob mice were randomly separated into two groups: DF (N  =  8) and SF (N  =  8). Each group continued on the designated feeding schedule for six weeks during which time body weights were taken regularly at ZT0 and ZT12 corresponding to food onset and offset. Caloric intake during the light and dark period was measured weekly by manual food removal.Ob/ob mice were randomly separated into two groups: DF (N  =  7) and SF (N  =  7). Before the onset of restricted feeding, mice were implanted with a leptin pump 24 hours prior to the first high-fat feeding period. Each group continued on the designated feeding schedule for two weeks during which time body weights were taken regularly at ZT0 and ZT12 corresponding to food onset and offset. Caloric intake during the light and dark period was measured weekly by manual food removal.Ob/ob mice were randomly separated into four groups: SF-SL (N  =  8), SF-DL (N  =  8), DF-SL (N  =  8) and DF-DL (N  =  8). Mice begin leptin injections 24 hours prior to the onset of restricted feeding. All groups continued on the designated feeding schedule for two weeks during which time body weights were taken regularly at ZT0 and ZT12 corresponding to food onset and offset. Caloric intake during the light and dark period was measured every 48 hours by manual food removal.To determine endogenous leptin levels , wild tyob/ob mice from Experiment 1 were then implanted with a leptin pump after six weeks of DF and then maintained on the DF feeding schedule for an additional two weeks. Body weights were measured daily for the first week and then every 2–3 days. The leptin pump was surgerically removed after two weeks and body weight gain was measured for an additional week after leptin removal.To determine if constant, non-rhythmic leptin leads to differential weight loss during DF , ob/ob mob/ob mice , a high-dose of leptin injection , or saline (N  =  4) over the course of one week. Body weights were measured once a day just prior to the leptin injection at ZT 11.To determine if the low-dose leptin injection (100 ug/kg/day) was sufficient to slow body weight gain in /ob mice , ob/ob mob/ob mice  or ZT 17 (N  =  6) for two weeks. Body weights were taken daily, 12-hours after the leptin injection. Plasma leptin was measured on the 11th day from 1-5 hours after the leptin injection.To determine the rate of clearance of the low-dose leptin injection in /ob mice  and if t/ob mice , ad libi