The 1.5-million-year-old Antarctic ice indicates a balanced atmospheric oxygen budget before the Mid-Pleistocene Transition. PO2) and the processes that act to regulate it remain enigmatic because of difficulties in quantitative reconstructions using indirect proxies. Here, we extend the ice-core record of PO2 using 1.5-million-year-old (Ma) discontinuous ice samples drilled from Allan Hills Blue Ice Area, East Antarctica. No statistically significant difference exists in PO2 between samples at 1.5 Ma and 810 thousand years (ka), suggesting that the Late-Pleistocene imbalance in O2 sources and sinks began around the time of the transition from 40- to 100-ka glacial cycles in the Mid-Pleistocene between ~1.2 Ma and 700 ka. The absence of a coeval secular increase in atmospheric CO2 over the past ~1 Ma requires negative feedback mechanisms such as Pco2-dependent silicate weathering. Fast processes must also act to suppress the immediate Pco2 increase because of the imbalance in O2 sinks over sources beginning in the Mid-Pleistocene.The history of atmospheric oxygen ( Past variations of O2 partial pressure (PO2) are commonly derived from geochemical models constrained by carbon and/or sulfur isotopes . For example, Landais et al.  over the past 800 thousand years (ka), but the authors did not provide a conclusive explanation on the origin of this decline .Ice cores offer a unique opportunity to study past variations of 2/N2, the magnitude of which is −8.4 ± 0.2‰/million year (Ma) (1σ)  also exhibit a persistent decreasing trend in δOPO2 to ~1.5 Ma using “snapshots” of δO2/N2 in the air trapped in the glacial ice from the Allan Hills Blue Ice Area (BIA). The data include samples first reported by Yan et al.  . Each individual δO2/N2 sample was assigned the age of the closest 40Aratm datum. Using this chronology, we divided samples into three distinct time bins: 1.5 Ma (± 0.1 Ma), 810 ka (± 100 ka), and 400 ka (± 70 ka). The numbers represent the average of 40Aratm ages weighted by their respective analytical uncertainty, bracketed by ±95% confidence intervals (CIs). The samples within each age bin do not necessarily have the exact same age. Rather, they are binned together because their age difference cannot be resolved by the existing 40Aratm dating method. This means that the true width of age distribution (data S1) is likely wider than the 95% CI shown in the brackets. This is to our advantage because integrating over longer time intervals filters out high-frequency variations in PO2 while preserving the long-term trend. Binning age groups this way allows the age uncertainty to be more rigorously defined and the effect of dating uncertainties on the long-term PO2 change to be quantified.Here, we extend the ice-core record of atmospheric PO2 from measurements of δO2/N2 of trapped air in ice cores requires corrections for fractionation during firnification and post-coring gas losses  of the snapshots could be biased toward periods with high accumulation rates/better sample preservation  in the trapped gases and use δAr/N2 as a proxy for fractionation modulated by insolation during firnification and fractionation due to post-coring gas loss.Reconstructing atmospheric 2 are fractionated during bubble close-off and gas loss in a fashion similar to O2 and N2 but different in magnitude, leading to the covariation between δO2/N2 and δAr/N2 in ice cores   and can also be neglected. Second, this method is expected to be robust to the partial preservation and recovery of the true range of δO2/N2 or δAr/N2 variability, as long as the slope of the δO2/N2-δAr/N2 data remains the same  between 120 and 700 ka to correct the δO2/N2 data measured from the same ice . Except in the youngest (135 ka) group, the corrected δO2/N2 falls almost exactly on the trend line of δO2/N2 versus time  is possible, although it is not firmly established from global deep-sea temperature records ] is therefore more likely to have contributed to the post-MPT δOPO2 due to changes in organic carbon burial and oxidation would inject CO2 to the coupled atmosphere-ocean system. Even if that decline is entirely due to the enhanced pyrite oxidation, atmospheric CO2 would still increase—albeit with a smaller magnitude due to interactions with the inorganic carbon cycle. When coupled with calcium carbonate dissolution, pyrite oxidation has a stoichiometric 8:15 ratio of CO2 produced to O2 consumed . Only when all the H+ produced during pyrite oxidation is consumed by silicate dissolution can Pco2 be truly decoupled from the oxygen cycle  enhanced CO2 storage in the deep Atlantic Ocean  is located ~100 km to the northwest of the McMurdo Dry Valleys in Victoria Land, East Antarctica. Sampling sites are located to the west of the Allan Hills nunatak in the Main Ice Field . Samples reported in this study come from two boreholes in the MIF with close proximity: ALHIC1502  and ALHIC1503 . Local glaciological conditions in the MIF are documented elsewhere (40Ar/38Ar ratio (40Aratm).While the glaciological conditions favor the preservation of old ice here, they also pose several challenges to using blue ice as paleoclimate archives. First, the unique glaciological dynamics at the BIAs make it difficult to constrain the chronology of the ice, as ice movement has likely disturbed the otherwise continuous ice stratigraphy. If the age of the blue ice samples is within 800 ka, they could be synchronized to the deep ice cores via various age markers  in the trapped gases  preclude an age assignment precise enough to establish a time series. To maximize the utility of the 40Aratm data, we classify samples into different age groups and calculate the average age (40Aratm measurements (ix), weighted by their corresponding uncertainty (σi)In addition, because the depths of δOThe error associated with the weighted mean is given by40Aratm. We acknowledge that there is no independent evidence to support this assumption. That being said, the purpose of such age binning is to provide a quantifiable age uncertainty that can then be taken into consideration in calculating long-term PO2 trend. Basing these calculations on the difference between the average age of two or more intervals has proven valid in EDC (fig. S3) and Vostok (fig. S4). In any case, uncertainties associated with age will affect the estimated rate of PO2 decline in the Late-Pleistocene, but will not change the conclusion of a stable atmospheric O2 level before the MPT.Underlying this error estimation is the implicit assumption that all samples within the age bin have the exact same age and the precision of age estimates is purely limited by the analytical precision of et al. (40Aratm of 400 ± 70 ka (95% CI) from 23 40Aratm measurements made on 20 depths in ALHIC1502 and ALHIC1503 cores. Similarly, the mean age of the MPT group is calculated to be 810 ± 100 ka (95% CI), constrained by nine 40Aratm data points from six depths. The pre-MPT age group has three sub-age units: 1.5, 2.0, and 2.7 Ma. In this study, we only include samples in the 1.5-Ma subgroup, the reason of which is discussed in the “Data quality and rejection criteria” section. This age  is estimated by four unreplicated 40Aratm.Three age groups were identified and listed in 40Aratm data that provide age constraints. The transition from post-MPT to MPT age unit in ALHIC1502 is located between 143.19 and 172 m, the last post-MPT and the first MPT 40Aratm-based age estimates, respectively. This data gap requires alternative estimates to identify the transition from post-MPT to MPT ice samples. By comparison, the chronology of ALHIC1503 is better constrained by 33 40Aratm analyses from 27 depths. We assume that ALHIC1502 and ALHIC1503 have an overall similar age-depth structure given their close proximity, and the variations in δ18Oatm can be used as a marker to identify the depth at which MPT ice first appears in ALHIC1502. In ALHIC1503, the transition from post-MPT to MPT age unit occurs along with a rapid δ18Oatm decline from 1.361 to 0.488‰  age in the deepest measured sample with a depth of 190 m. In contrast, the deepest 40Aratm measurement in ALHIC1503 yields an age of 2.7 ± 0.3 (1σ) Ma. There are two possible explanations: (i) The ice dates back to the MPT and is buried under the pre-MPT ice due to stratigraphic disturbance or (ii) the age is underestimated because of input of radiogenic 40Ar from the bedrock. We have no way of identifying the true cause(s), but either way, it would not affect the conclusion of this paper because the δO2/N2 data were rejected on other bases .Another interesting feature of the depth profile of 2/N2, δAr/N2, and δ15N of N2 data reported in this study are modified from Dreyfus et al.  to mass 44 (12C16O2), thereby yielding raw 15N/14N, O2/N2, Ar/N2, and 18O/16O ratios at the same time. Analysis of each single sample was made up of 24 individual cycles, each with a 16-s integration time. The raw δ15N, δ18O, δO2/N2, and δAr/N2 ratios were corrected for pressure imbalance based on the ion currents on the sample and reference sides  . Those gravitationally corrected data, presented in data S2, are used for interpreting atmospheric O2 concentrations.The dip tube was allowed to warm up at room temperature for at least an hour. Later, it was attached to an isotope-ratio mass spectrometer (Thermo Delta Plus XP) for elemental and isotopic analysis for ~30 min. The configuration of the mass spectrometer permits simultaneous measurement of mass 28  dating back to 1.5 Ma as an example. With a pooled SD of ±3.372‰ (1σ), 25 unique samples each with replication should yield a 95% CI of ±0.95‰, if all the disagreements between the δO2/N2 replicate values arise from the mass spectrometer measurements. The calculation is as follows: The observed pooled SD (3.372‰) is first divided by the square root of 2 to take replicate analyses into account, then divided by the square root of 25, the number of unique samples, and finally multiplied by 2 to yield the 95% CI. However, the 95% CI of the δO2/N2-δAr/N2 regression line is ±0.79‰ at δAr/N2 = −7.1‰, less uncertain than what pure analytical uncertainties alone would dictate. This observation means that correction by δAr/N2 reduces the uncertainties in the δO2/N2 dataset. Thus, the large analytical uncertainty of δO2/N2 measured on ice with bubbles must partly be sourced from processes that fractionate both δO2/N2 and δAr/N2, such as the different extent of gas losses experienced by the replicates during handling. The CI of the regression line and the estimated δO2/N2 can certainly benefit from improved analytical precision in the future.Here, we quantitatively investigate how much the observed δO2/N2, we used a “York Fit” algorithm that takes into account the inherent uncertainty in both the independent (δAr/N2) and dependent (δO2/N2) variables in a generalized linear model  , the York Fit yields a steeper slope, but none is significantly different from the slope yielded by the ordinary least squares (OLS) regression method used in our analysis. For the post-MPT samples, the York Fit slope is 2.16 ± 0.84  with a y intercept of 1.9 ± 5.9‰, compared to the slope of 1.74 ± 0.44 and the y intercept of −0.9 ± 3.1‰ yielded by the OLS regression. For the MPT samples, the York Fit slope and y intercept are 2.31 ± 1.40 and 7.4 ± 10.6‰, respectively, while the OLS regression slope and y intercept are 1.60 ± 0.50 and 2.2 ± 3.8‰, respectively. The MPT samples have the largest departure of York Fit from the OLS fit. Last, for the pre-MPT samples, a slope of 1.77 ± 1.00 and a y intercept of 3.9 ± 7.5‰ are obtained from the York Fit, whereas the OLS slope and y intercept are 1.51 ± 0.38 and 2.0 ± 2.9‰, respectively.To investigate how large analytical uncertainties could potentially affect the estimate of δO2/N2 when δAr/N2 is normalized to −7.1‰ does not change much (table S1), visually represented by the two types of regression lines intersecting near δAr/N2 = −7.1‰ (fig. S6). The corrected δO2/N2 (± 2σ) from York Fit is −13.47 ± 1.68, −8.96 ± 1.04, and −8.69 ± 1.06 for post-MPT, MPT, and pre-MPT samples, respectively. These results are very close to the OLS estimates made in the main text (table S1), because the value of −7.1‰  is very close to the mean values of δAr/N2 in each age group. Visually, the York Fit tilts the slope of OLS regression around the arithmetic mean of the independent variables (δAr/N2). The estimated δO2/N2 near the arithmetic mean of δAr/N2 is therefore not substantially changed, but extrapolating δO2/N2 to δAr/N2 = 0 would lead to large differences (table S1). For these reasons, we still chose to regress δO2/N2 to δAr/N2 = −7.1‰ using the OLS method while acknowledging its inadequacy in addressing uncertainties in the variables. This facilitates the comparison of our regression results with the slopes of earlier work, all of which were done using the OLS method without factoring in the uncertainties in δO2/N2 or δAr/N2.Despite the changes to the slope when analytical uncertainties are taken into account, the estimated δO2/N2 and δAr/N2 dataset measured on ice collected from the Allan Hills BIA. The first batch was measured in 2013–2014 on samples drilled in 2010–2011 Antarctic field season. This batch of data (“batch 1”) was first reported by Higgins et al. .2/N2 and δAr/N2 data reported in the present study come exclusively from ice cores drilled in the 2015–2016 field season. They have been stored at −36°C in the National Ice Core Facility in Denver, CO until retrieval for cutting and processing shortly before laboratory analyses and have no surface cracks or fractures. These storage and procedural differences may help explain the larger scatter observed in the δO2/N2-δAr/N2 data reported by Higgins et al. . The δO2/N2 associated with this sample is −26.3‰, substantially (>5‰) more negative than typical ice core values. These anomalous negative ratios may arise from considerable gas loss experienced by this particular sample that greatly depletes Ar and O2 relative to N2. Six additional post-MPT samples were rejected because they are bracketed by pre-MPT age bins and might represent a different time slice than the shallow post-MPT samples. We still list these samples in data S2 but mark them as “anomalous gas loss” and “stratigraphy,” respectively. Including these data points in the regression analysis does not change the conclusion of this paper.One post-MPT sample was rejected from batches 2 and 3 because of its very negative δAr/N2 consumption does not occur in situ inside the ice being measured, the large O2 gradient could potentially drive a diffusive flux and modify the ice core δO2/N2. Many 2.0-Ma and all 2.7-Ma samples come from the sections are affected by respiration, evidenced by the depleted δ13C values and anomalously elevated CO2 levels . There are only four 2.0-Ma samples that are considered “pristine” . These four pristine δO2/N2-δAr/N2 data points fall within the envelop defined by the rest of the Allan Hills blue ice δO2/N2-δAr/N2 data, but they were excluded from inferring Early-Pleistocene PO2 levels because of the small number of samples (annotated “2 Ma samples” in data S2). O2 consumption by nonmicrobial redox reactions such as Fe(II) oxidation has been observed in the nearby (~500 km) Talos Dome ice core  during glacial and <15 ppb during interglacial periods]  in the basal EDC ice below 3200 m  was the reason, the 60-m layer would need to span an unlikely ~10 ka in time  for calculations performed in this study.


Throughout the nervous system, the organization of excitatory and inhibitory synaptic inputs within a neuron’s receptive field shapes its output computation. In some cases, multiple motifs of synaptic organization can contribute to a single computation. Here, we compare two of these mechanisms performed by two morphologically distinct retinal direction-selective ganglion cells (DSGCs): directionally tuned inhibition and spatially offset inhibition. Using drifting stimuli, we found that DSGCs that have asymmetric dendrites exhibited stronger directionally tuned inhibition than symmetric DSGCs. Using stationary stimuli to map receptive fields, we found that DSGCs with both symmetric and asymmetric dendrites exhibited similar spatially offset inhibition. Interestingly, we observed that excitatory and inhibitory synapses for both cell types were locally correlated in strength. This result indicates that in the mouse retina, dendritic morphology influences the amount of tuned inhibition attained through asymmetric wiring but does not dictate the synaptic organization of excitation relative to inhibition. Neural circuit function is dependent on the detailed organization of excitatory and inhibitory synapses onto dendrites. Here, we use a classic neural circuit, the direction-selective circuit of the retina, to assess how changes in dendritic shape impact the synaptic organization. We find the direction-selective cells of the retina that have asymmetric dendrites have similar synaptic organization to those that have symmetric dendrites, indicating the shape of dendrites does not dictate the final computation of the neurons.Detecting the direction of moving stimuli is an essential part of sensory processing. In the mouse visual system, direction selectivity is first observed in the retina, where direction-selective ganglion cells (DSGCs) fire many action potentials in response to motion in their preferred direction, and few to no action potentials in response to the opposite, or null, direction. Direction-selective computations occur across many layers of the mammalian visual system from DSGCs of the retina, to the retino-recipient neurons of the dorsal lateral geniculate nucleus (dLGN) of the thalamus , thalamoRetinal direction selectivity is mediated primarily by inhibition through two non-mutually exclusive mechanisms. The first mechanism is based on directional tuning of inhibition, where the amount of inhibitory input onto a DSGC is greater for null direction motion than for preferred direction motion. In the mammalian retina, this tuned inhibition is provided by starburst amacrine cells (SACs), where the combination of SAC centrifugal directional tuning , and DSGThe second mechanism is based on spatially offset inhibition, a term used to describe when excitatory and inhibitory receptive fields are spatially offset from each other. Hence, during preferred direction motion, the stimulus elicits an excitatory response before an inhibitory response, allowing the cell the fire action potentials. During null direction motion, the stimulus elicits an inhibitory response primarily at the same time as the inhibitory response, effectively suppressing spiking output. This is the classic mechanism postulated to underlie direction-selective responses in both the retina  and in tA receptors on their dendrites  to p60 and were of both sexes. Animals used in experiments had not previously been involved in other experiments or exposed to any drugs. Animal health was monitored daily and only healthy animals were used in experiments. To target vDSGCs, we used Hb9::GFP  mice, wh2/5% CO2) Ames’ media (Sigma) for light responses. Retinal orientation was determined as described previously (2 hole in nitrocellulose filter paper (Millipore) with the photoreceptor layer side down and stored in oxygenated Ames’ media until use (maximum 10 h). All experiments were performed on retinas in which dorsal-ventral orientation was tracked.Mice were anesthetized with isoflurane and decapitated. Retinas were dissected from enucleated eyes in oxygenated  were generated using a computer running 420- to 520-nm light through a digital micro-mirror device (DLI Cel5500) projector with a light emitting diode (LED) light source generated using MATLAB software with the Psychophysics Toolbox. Visual stimuli are focused on the photoreceptor layer using a condenser in the DMD path to the chamber.To measure the directional tuning of synaptic currents onto DSGCs, drifting bars of positive contrast on a gray background (96% Michaelson’s contrast) were presented  in eight block shuffled directions, repeated three times, moving along the long axis of the bar. Each presentation lasted 6 s and was followed by 3 s interstimulus interval of gray background. For these moving stimuli, the illumination radius on the retina was 1.4 mm to limit modulation of DSGC responses by inhibitory wide-field amacrine cells . A 20× w5 R*/s/rod. Stimuli were individually presented in 100 block-shuffled positions, repeated three times, with each stimulus lasting for 0.5 s followed by a 1.2 s interstimulus interval of gray background. Stimuli were presented within a 10 × 10 grid, onto a stimulus field of 500 × 500 μm, with the DSGC soma located in the center of the stimulus field.To map excitatory and inhibitory receptive fields of DSGCs, positive contrast square stimuli (30 × 30 μm) were flashed over a gray background (96% Michaelson’s contrast) at an intensity of 3.1 × 10m CsMeSO4, 2.8 mm NaCl, 20 mm HEPES, 4 mm EGTA, 5 mm TEA-Cl, 4 mm Mg-ATP, 0.3 mm Na3GTP, 10 mm Na2phosphocreatine, and 5 mm QX-Cl . Whole cell recordings were performed with the same pipette after obtaining a giga ohm (1 GΩ) seal and breaking into the cell membrane. Holding voltages for measuring excitation and inhibition after correction for the liquid junction potential (10 mV) were 0 and −70 mV, respectively. Signals were acquired using Clampex 10.4 recording software and a Multiclamp 700A amplifier (Molecular Devices), sampled at 10 kHz, and low pass filtered at 6 kHz.Oriented retinas were placed under the microscope in oxygenated Ames’ medium at 32–34°C. Identification and recordings from GFP+ cells were performed as described previously . In brieAfter physiological recordings of DSGCs were completed, Alexa Fluor 594-filled DSGCs were imaged using two-photon excitation at 800 nm. At this wavelength, GFP is not efficiently excited, but Alexa Fluor 594 is brightly fluorescent; 480 × 480 μm Image stacks were acquired at z intervals of 1.0 μm and resampled fifteen times for each stack using a 20× objective  30 kHz resonance scanning mirrors covering the entire dendritic fields of the DSGCs. Image stacks of DSGCs were then imported to FIJI (NIH) and a custom macro was used to segment ON and OFF dendrites based on their lamination depth in the inner plexiform layer . Following ON and OFF dendritic segmentation, we used the Simple Neurite Tracer plugin in FIJI to skeletonize and then binarize the ON and OFF dendritic segments for morphologic analyses.m in Ames’ media, and allowed it to perfuse for 5–10 min at a perfusion rate of 1 ml/min.For experiments conducted in Hexamethonium (Millipore Sigma), we diluted 100 μp values are indicated in the figures and figure captions; p < 0.05 was considered significant.Mean ± SDs for all angles performed using circular mean and circular SDs. Details of statistical tests, number of replicates, and For voltage clamp recordings during moving stimuli, traces were first averaged across the three trials for each direction and inspected to ensure the consistency of the responses across trials. Average traces were baseline subtracted based on the last 500 ms of recording or a user defined interval after manual inspection. Peak currents were calculated from average baseline subtracted traces. They were defined as the maximal (IPSC) or minimal (EPSC) points during two separate 1.9 s windows in which the ON and OFF responses occurred. The peak currents in each direction were used to calculate the vector sum of the current responses. For timing analysis, PSC traces were low pass filtered using an 80-ms moving average, and the peak times for excitation and inhibition was extracted for both ON and OFF responses. Null directions for both ON and OFF responses were defined as the angle of the vector sum of ON and OFF peak IPSCs; the preferred directions were defined as 180° from the, null direction.The directionally selective index (DSI) was calculated for the peak amplitude of the IPSCs as: (ND – PD)/(ND + PD), where ND is the amplitude of the peak current in the null direction, and PD is the amplitude of the peak current in the preferred direction. We also used the magnitude of the vector sum of the PSCs as another measurement of directional tuning  of the receptive field using the following equations:x and y are the cartesian coordinates of the COM, N is the total number of stimulus squares (100), m is the peak current amplitude at each coordinate location, and M is the sum of peak current amplitudes across the entire receptive field.Where To measure the displacement and orientation of the receptive fields relative to the soma, we calculated the magnitude and angle, respectively, of vector from the soma to the COM of the receptive field using the following equations:x and COMy, respectively.Vector magnitude = To quantify spatially offset inhibition, we calculated the vector from the COM of the excitatory receptive field to the COM of the inhibitory receptive field.To compare DSGC dendrites to the synaptic inputs evoked by static stimuli, we skeletonized dendrites using methods described above. Next, we calculated the vector from soma to the COM of the dendritic pixels; the magnitude of the vector indicates the magnitude of dendritic asymmetry relative to the soma, while the angle of the vector indicates the orientation dendrites. To directly compare the DSGC dendrites to the IPSC and EPSC receptive fields, we binned the dendritic skeleton into a 10 × 10 matrix by summing the binarized pixels in each bin, with the soma located in the center of the matrix. Again, we calculated the vector from soma to the COM of the binned dendritic pixels; the magnitude of the vector indicates the magnitude of dendritic asymmetry relative to the soma, while the angle of the vector indicates the orientation dendrites. In To quantify receptive field size, the locations in the excitatory and inhibitory pixels that had responses below a set noise threshold of 50 pA were assigned a value of 1 while the other pixels were assigned a value of zero. Next, we calculated the area total area of the grid with responses that exceed the threshold. To quantify dendritic field size in a manner that is comparable to the receptive field size, we binned dendritic pixels into a 10 × 10 matrix and then used the same method to calculate dendritic area, without applying a threshold since dendrites were skeletonized before this analysis using the method described above.Our goal was to determine if the synaptic organization onto DSGCs is dependent on the dendritic morphology. We first quantified the difference in dendritic asymmetry in vDSGCs versus nDSGCs by calculating the magnitude of the vector from the soma to the COM of the dendritic pixels. We found that both ON and OFF dendrites of vDSGCs were significantly more asymmetric than nDSGCs . As repoTo assess the impact of dendrite morphology on the tuning of inhibition, we conducted voltage clamp recordings of both vDSGCs and nDSGCs and isolated IPSCs in response to a bar of light moving in eight different directions B. DespitPrevious studies have reported differences in the relative timing of excitatory and inhibitory synaptic inputs for preferred and null direction stimulation, consistent with the presence of spatially offset inhibition . Here, wPreviously, we showed that in asymmetric vDSGCs, the centers of mass of the spatial receptive fields for excitation and inhibition are both offset toward the preferred direction with inhibitory receptive fields further offset than the excitatory receptive fields . HoweverTo characterize the relative position of excitatory and inhibitory receptive fields, we computed the COM for dendrites, excitatory receptive fields, and inhibitory receptive fields B and comAlthough we observed a shift in the position of inhibitory receptive fields relative to excitatory receptive fields in both cell types, there was also a striking correlation between them. First, we observed a strong positive correlation between the location of excitation and inhibition relative to the soma F. Note, To assess the organization of the excitatory and inhibitory receptive fields along the preferred-null axis, we collapsed the synaptic currents recorded with the static stimulus along the axis orthogonal to their preferred direction and plotted the normalized distribution of excitation and inhibition A. We fouWe next explored whether the small displacements for the EPSC and IPSC receptive field centers from the soma were correlated with variations in the spatial arrangement of the DSGC dendrites A. To do In the above experiments, EPSCs are mediated by a combination of activation of nicotinic acetylcholine receptors (nAChRs) and glutamate receptors. In a subset of experiments, where we pharmacologically blocked cholinergic excitation, we found that the orientation of the glutamate receptive field in vDSGCs was also ventrally oriented Dendritic morphology is thought to influence synaptic organization. Here, we show that dendritic morphology impacts the amount of tuned inhibition whereby DSGCs with asymmetric dendrites exhibit more strongly tuned inhibitory inputs than DSGCs with symmetric dendrites but both cell types exhibit comparable spatially offset inhibition. Moreover, in both cell types, we found that the receptive fields for excitation and inhibition were similarly oriented to each other and were locally correlated in strength. Finally, our results indicate that spatial receptive fields are significantly larger than dendritic fields and are not strongly dictated by the dendritic structure. Here, we discuss the implications of these findings for direction selectivity in the mouse retina.Drosophila, where connectome analysis reveals dendritic asymmetry mediates the physical displacement of null and preferred side inputs (We found that vDSGCs had stronger inhibitory tuning than symmetric nDSGCs, driven primarily by a decrease in the amount of inhibition during preferred side stimulation . One inte inputs . Anothere inputs .Despite the difference in tuning of inhibition, vDSGCs and nDSGCs have been shown to exhibit similar spike tuning properties under our stimulus conditions . We thinAn alternative interpretation is that different subtypes of DSGCs receive different levels of non-DS inhibition from other sources  such as Using receptive field mapping based on stationary stimuli, we find that there was an overall shift in the inhibitory receptive field relative to the excitatory receptive field for DSGCs with both symmetric and asymmetric dendrites. This is consistent with our previous work which has shown that vDSGCs exhibit ventrally offset inhibitory receptive fields, regardless of their altered morphology following dark rearing .In our present study, the spatial offset between the excitatory and inhibitory receptive fields was on average <50 μm E, which We also found that both excitatory and inhibitory receptive fields were much larger than dendritic fields . BlockadIt is important to note that the strength of synapses revealed by stationary receptive field mapping is different from what is activated during moving stimuli. Motion stimuli evoke directional release of GABA from SACs , and gluHere, we find that the while the glutamatergic receptive fields of nDSGCs are skewed toward the DSGC’s null direction, consistent with previous reports , the gluAs noted above, the extent of direction-selective tuning is set by the presence of a sufficient level of inhibition. Interestingly, there is strong evidence that the direction-selective computation is made locally on DSGC dendrites, i.e., that motion stimuli confined to small segments of the DSGC dendritic tree still elicit a directional responses . First, Computational modeling showed that nonlinear conductance within the dendritic tree promotes a multicompartmental model, allowing local interactions between excitation and inhibition to shape dendritic DS, while SAC ablation abolished DS . A multiIn conclusion, we show that DSGCs exhibit two parallel mechanisms for computing motion direction. The first is based on tuned inhibition, which we find is influenced by the morphology of the DSGC, and the second is based on spatially offset inhibition which is not influenced by the DSGCs’ dendritic orientation, size or asymmetry.


In select DSP variants, disease is linked specifically to the uncovering of a previously-occluded calpain target site (residues 447–451). Here, we partially stabilize these calpain-sensitive DSP clinical variants through the addition of a secondary single point mutation—tyrosine for leucine at amino acid position 518 (L518Y). Molecular dynamic (MD) simulations and enzymatic assays reveal that this stabilizing mutation partially blocks access to the calpain target site, resulting in restored DSP protein levels. This ‘molecular band-aid’ provides a novel way to maintain DSP protein levels, which may lead to new strategies for treating this subset of DSP-related disorders.Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, a subcellular complex that links the cytoskeleton of one cell to its neighbor. A mutation ‘hot-spot’ within the NH Arrhythmogenic cardiomyopathy (ACM/ARVC), one of the cardiomyopathies associated with desmoplakin hotspot variants, is a rare 1:2000–1:5000) inheritable disease ,4,5. Rai000 inherEighty percent of ACM cases are linked to variants in genes that encode desmosomal proteins . The desDesmoplakin (DSP) is an essential protein within the desmosome ,14. WithAround 5% of ACM cases are linked to DSP variants ,13,16. MCalpain is a ubiquitously expressed calcium-activated cysteine protease . This prDSP variants produce otherwise functional proteins that are prematurely degraded by calpain, one obvious solution is to block calpain cleavage using calpain inhibitors ). Wildtype DSP was induced with IPTG at an OD600 of 0.6 and grown for an additional 4 h at 30 °C. Mutant constructs were induced for 10 h at 16 °C. Cells were pelleted, resuspended in phosphate buffered saline (PBS) with the Complete Protease Inhibitor (Roche), sonicated and centrifuged. The supernatant was incubated with glutathione sepharose, and protein was eluted using a 10 mM reduced L-glutathione PBS buffer solution. Elution containing protein was collected, concentrated, and applied to a S200 size chromatography column. Protein purity was determined by SDS-PAGE.Circular Dichroism (CD). CD spectra were obtained using a 3-mm quartz cuvette in a JASCO J-1500 spectrometer in 5 °C increments from 15 °C to 90 °C. Experiments were completed in triplicate, using 1–3 μM protein in PBS, pH 8.0. Spectra were taken from 200 to 300 nm.Calpain assays. Calpain assays were performed as described in [2 and calpain, for a final DSP concentration of 9.97 μM. Reactions were incubated at 37 °C for 30 min, quenched, and analyzed by SDS-PAGE followed by staining with Sypro Ruby (ThermoFisher) total protein stain according to the manufacturer’s instructions. Quantification of the total amount of protein remaining was calculated via densitometry obtained from ImageJ software (NIH). Values for at least 4 independent replicates were averaged for each variant.ribed in  with minStatistics.Data are represented by the mean and SEM. Multiple comparisons were done with 1-way ANOVA and a Tukey HSD test was used to determine statistical significance. In addition, p values of less than 0.05 were considered significant.Molecular Dynamics. Computational modeling was done using the human wildtype DSP residues 178–627 (PDB accession 3R6N) [on 3R6N) . S299R, on 3R6N) ; free_saon 3R6N) ). Calculon 3R6N) , and daton 3R6N) .2-terminal third of the protein is composed of 6 α-helical spectrin repeats (SRs) and a Src homology 3 (SH3) domain important in tethering DSP to the desmosomal complex [2-terminal third of the molecule, within ~15 Å of the SH3/SR4 interface [DSP is a modular protein with three distinct regions in both structure and function. The NH complex ,38. The  complex . Four ACnterface ,17. ThesIt is essential to consider several factors when designing a possible “rescue” mutation for disrupting the calpain targeting of DSP. Calpain targeting efficiency is influenced by amino acid type up to seven residues away from the scissile bond ; therefoTo determine whether the introduction of L518Y blocks DSP targeting by calpain effectively, we performed calpain cleavage assays on recombinant DSP (amino acids 1-883) with and without clinical variants . ResultsThe L518Y mutation partially protects DSP variants in both the presence .The MD simulation analysis of each DSP variant reveals that the secondary “rescue” L518Y mutation hides the calpain target site by decreasing its exposed surface area, as predicted. This occlusion is evident in all variants except S299R, which already has a smaller exposed surface area compared to its L518Y double mutant  Column 6r trends . The sur2) to completely cover the fully exposed calpain target site (~200–300 Å2). Additionally, the simulations reveal that L518Y is positioned over the calpain target site only about 30% of the time (o) [While a decreased target site exposure brought about by L518Y provides a logical explanation for the observed DSP clinical variant rescue, we had originally hypothesized that this protection would be quite robust. To probe the mechanism of why the presence of L518Y offers only relatively weak protection against DSP proteolysis, we examined the orientation of L518Y in MD simulations more closely. First, we noted that one face of tyrosine itself is too small (~60 Åthe time C. This ptime (o) . In hindWe next examined why L518Y protects the distal variants from degradation more fully than the proximal variants. The calpain target site resides in a loop between the SH3 domain and the beginning of SR5 and is stabilized on three faces by intramolecular interactions A. The L5To explore the proximal vs. distal variant stability effects more thoroughly, we examined all noncovalent interactions stabilizing the calpain target site (residues 446–452) B. The diAn in-depth analysis of the calpain target site also revealed that in addition to physically occluding one side of the target site, L518Y also stabilizes the proximal but not the distant mutant structure . TherefoHere we provided evidence for a proof-of-concept that physically blocking a calpain target site can rescue DSP levels. We have created a partial “molecular band-aid” in the form of L518Y, a mutation which can impede calpain targeting of DSP. However, careful analysis shows that this band-aid is only partially functional due to its size, position, and mobility. It follows that to fully protect the calpain target site, it will be necessary to develop large molecules or antibodies that bind to larger patches of the exposed surface of DSP. Keeping both the caveats and the successes identified here in mind should aid in creating more effective future compounds that block DSP degradation.


Surface meltwater reaching the base of the Greenland Ice Sheet transits through drainage networks, modulating the flow of the ice sheet. Dye and gas-tracing studies conducted in the western margin sector of the ice sheet have directly observed drainage efficiency to evolve seasonally along the drainage pathway. However, the local evolution of drainage systems further inland, where ice thicknesses exceed 1000 m, remains largely unknown. Here, we infer drainage system transmissivity based on surface uplift relaxation following rapid lake drainage events. Combining field observations of five lake drainage events with a mathematical model and laboratory experiments, we show that the surface uplift decreases exponentially with time, as the water in the blister formed beneath the drained lake permeates through the subglacial drainage system. This deflation obeys a universal relaxation law with a timescale that reveals hydraulic transmissivity and indicates a two-order-of-magnitude increase in subglacial transmissivity (from 0.8 ± 0.3  Hydraulic transmissivity under the 1km-thick Greenland Ice Sheet was inferred by ice-sheet uplift relaxation after rapid lake drainage events. A two-order-of-magnitude increase in hydraulic transmissivity was found throughout the melt season. Surface meltwater entering the subglacial drainage system at the ice-bed interface plays crucial roles in modulating the flow of the ice sheet7. As the melt season progresses, observations and theories suggest that the basal hydrologic system beneath the ice sheet transitions from an inefficient distributed network to a more efficient channelized system that transmits more water at lower pressure11. Drainage type, whether distributed or channelized, impacts basal sliding and ice discharge10.Thousands of supraglacial lakes form annually on the surface of the Greenland Ice Sheet in response to seasonal melt that fills topographic depressions. Many of these lakes drain rapidly (<1 day), transporting meltwater through vertical hydrofractures directly to the ice-bed interface11 suggest a progressive channelization throughout the melt season, and indicate that a 5-fold increase in subglacial water speed occurs as a channelized system develops near the terminus10. However, the effect of channelization tapers further inland12 at higher elevations (roughly > 1000 m above sea level [a.s.l.])14, where evolution of local basal hydrology remains poorly constrained by observations. Furthermore, observational-based estimates for hydraulic transmissivity, a key parameter controlling the water discharge in a subglacial sheet for a given hydraulic potential gradient, are scarce. Here we calculate the local hydraulic transmissivity15 beneath rapidly draining supraglacial lakes (1000–1350 m a.s.l.) at their time of drainage using a combination of field observations, a mathematical model, and laboratory experiments in order to quantify the seasonal changes of local hydraulic transmissivity driven by seasonal meltwater inputs.Understanding the degree and the spatial extent of the drainage system transition is paramount for characterizing the relationship between seasonal ice flow perturbations and surface melt. Field-based tracer studies conducted in the western margin of the ice sheet16. After a lake drainage event, surface uplift decays over time1 as water exits the blister and spreads along the ice–bed interface  of the ice sheet after a lake drains to quantify the hydraulic transmissivity15 of the surrounding subglacial drainage system.The rapid drainage of lakes injects surface meltwater into the ice–bed interface. The water can be transiently stored in water-filled “blisters” Fig.  that liface Fig. . PreviouUsing ice-sheet surface elevation data from on-ice GPS stations, we characterize the relaxation timescale for five lake drainage events that occurred between 2006 and 2012 at three separate lakes located up to ~200 km apart along the western margin of the Greenland Ice Sheet Fig. . Ice-she011 Fig. ) and sho006 Fig. ).Relaxation of surface uplift following a lake drainage event can be explained by gradual drainage of the blister into the neighboring subglacial drainage system Fig. . Based o11 from an inefficient, distributed drainage system early in the melt season to an efficient, channelized system later in the melt season. However, at elevations where supraglacial lakes form (~1000 m a.s.l.), field evidence suggests that channels do not develop until early August10. Subglacial hydrological models indicate that short, discontinuous channels form following lake drainage, but the discharge from the channel is small compared to the discharge through the distributed sheet13. In reality, either a purely distributed system without any channels or a channelized system dominated by turbulent discharge are unlikely end members of the subglacial drainage system beneath the lakes. Therefore, instead of quantifying the efficiency of a purely distributed sheet or a purely turbulent channel, here we use transmissivity to characterize the bulk subglacial drainage system , where a wide range of drainage features likely co-exist . A similar approach was developed by Sommers et al. 24. Because the horizontal extent of the water flow (~kilometers) is much larger than the vertical extent (~meters), the bulk subglacial drainage system is treated as a continuum water sheet of effective depth Near the ice-sheet margin the subglacial drainage system is thought to evolve seasonallyUplift relaxation is driven by the elastic deformation of ice lying above the water-filled blister and resisted by the viscous dissipation of the water flow in the distributed drainage system. Below we use scaling arguments to obtain a characteristic timescale of blister relaxation mentclass2pt{minimFor a blister of maximum thickness t}R Fig.  the blisequation , where \22, where the water flux 13, Darcy’s law holds to the first order. Thus, the bulk subglacial flow obeys Darcy’s law The flow in the bulk subglacial water sheet can be described by Darcy’s law for flow in porous materialsion Fig.  and is aequation  of magni27, the magnitude of the elastic stresses in ice can be estimated by Hooke’s law corresponding to an approximate strain Deformation of the ice overlying the blister generates elastic stresses in the ice sheet. For a penny-shaped blisterequation :3\documeFor a typical blister radius entclass1pt{minimaThe relaxation dynamics are governed by a balance between the elastic stresses mentclass2pt{minimus, Eqs.  give theequation  that do This timescale will be used to rescale the experimental and field data.Considering mass conservation and a force balance analysis  is show28. In contrast, here we consider a porous substrate that mimics the drainage system. In our experiment, a fluid-filled blister 16. Our lab experiments show that varying the permeability of the porous substrate 29.Next, we tested the analytic model (Eqs.  and 6)))6)) againts Fig. . The blition , the ext}tc  but also effective thickness We then applied the validated model to estimate hydraulic transmissivity mentclass2pt{minimmentclass2pt{minimequation ; Table 1To demonstrate the universality of the time-dependent uplift relaxation data, we rescaled the surface uplift data Fig.  by the cfrom Eq.  using thpse Fig.  onto thepse Fig.  Thus, deata Fig.  and dimeata Fig.  fall ontata Fig. ), demons3). By contrast, the mid-season events relax faster and are best explained by high transmissivity (3). When comparing our estimates of hydraulic transmissivity to modeled cumulative surface runoff31 at the time of lake drainage, we observe that transmissivity generally increases with increased cumulative runoff 32 included a simple linear relationship between melt input and hydraulic conductivity in a subglacial hydrological model and found an improved match between the modeled subglacial water pressure with borehole observations in the late melt season. Our work provides observational evidence of the seasonal changes of hydraulic transmissivity. Note that our result does not imply that transmissivity solely depends on cumulative surface runoff. The processes controlling the shift in transmissivity are left for future studies. Comparing the range of transmissivity values we obtained from the field data (3) with the hydraulic conductivity used in large-scale hydrological models13 (−1), we calculate that the sheet depth is of order Finally, we compare the transmissivities for lakes draining during the early- and mid-melt season. Our results suggest the local transmissivity of the basal drainage system beneath North Lake can change by up to two orders of magnitude between early June and late July. Early-season events relax more slowly as characterized by a low-transmissivity drainage system , however, the measured water velocity must be averaged along the entire tracer pathway 10 gives a water velocity averaged along a 41-km-long water pathway). Thus, it is unclear to what extent the drainage system evolves locally in higher elevation regions from dye and gas tracer experiments. Our results imply that the drainage system likely evolves locally at the lake elevations (~1000 m a.s.l.). In addition, tracer experiments are limited to sampling moulins in land-terminating glaciers located mainly in south-west Greenland. By contrast, supraglacial lakes are widespread over the ice sheet and are forming in progressively higher elevation regions as the climate warms33.We infer that local hydraulic transmissivity under North Lake (~1000 m a.s.l.) likely changes seasonally. Our method can be applied to estimate local transmissivity beneath other draining supraglacial lakes simply based on surface observation of uplift and lake volume. Dye and gas tracer experiments34, total melt season runoff at a 1500 m a.s.l. location up-flowline of North Lake in 2100 is projected to be higher than the average total melt season runoff from 2006 to 2012 observed at North Lake (~1000 m a.s.l.)  is crucial for understanding ice-sheet behavior in a warming climate. Our method provides an approach to determine the magnitude of transmissivity of high-elevation regions using only observations of surface deformation and lake volume. Under RCP scenario 8.5l.) Fig. . Thus, o38, following the methodology previously presented for the same GPS data in Stevens et al. 2 and Stevens et al. 39. GPS data for each station were processed individually relative to the 30-s resolution Greenland GPS Network (GNET) KAGA base station located on bedrock, 55 km from North Lake40. 1-2. GPS data are archived at UNAVCO .For the 2006, 2011, and 2012 North Lake and 2009 South Lake drainage events, continuous 30-s resolution GPS data collected by dual-frequency Trimble NetR9 receivers were processed with Track software2, thus the data during the first peak is avoided in the fitting of the linear model. The uplift data for Lake F   Digital Elevation Model (DEM) from GeoEye and WorldView Imagery, Version 13, with maximum lake depths on the order of 10–15 m , given the GIMP DEM time stamp is in 2012, and the last pre-drainage image of South Lake was taken 4 days before the 2009 drainage. Following this methodology, we estimate the 2009 South Lake drainage volume to be 0.03 ± 0.01 km3.Based on a comparison between the mapped South Lake margin and GIMP DEM elevation contours, the South Lake margin elevation was estimated to be 1050.5 m a.s.l. 2, which solves for the blister height based on surface displacement data from a network of 15 GPS stations around the blister at North Lake. We would expect the uncertainties in The maximum blister height entclass1pt{minimaequation . We thenentclass1pt{minimaSince blister volume is equation , the blientclass1pt{minimaFinally, the errors associated with estimating the transmissivity entclass1pt{minimaOne benefit of dimensionless solutions is that the shape of uplift data lot Fig. . Theoretlot Fig. ) dependsequation . We estimentclass2pt{minimThere are two constants that can cause systematic uncertainties: the Young’s modulus of ice  model’s monthly output averaged from 2006 to 2012  175 in 2011), roughly 6 days after the initial uplift peak on 2011/169. There are three LandSat-7 images available over this time window , we observed the time evolution of the fluid in the porous substrate and the blister , which mimics ice, to a porous substrate (PDMS micropillar array), which serves as a simple model for the porous drainage system Fig. , using ater Fig. . Darker The uncertainties associated with experimental parameters are listed in Supplementary Table A porous substrate made of micropillar arrays is used to model the porous drainage system. We designed and fabricated silicon molds for the micropillar arrays using standard photolithography methods. Each mold for the micropillar arrays is 7 cm in diameter and consists of circular wells on a hexagonal array with a porosity of 50%. Three variations of the micropillar array molds were designed with well diameters of 500 μm, 250 μm, and 125 μm and a well depth of 90 μm for each. Polydimethylsiloxane (PDMS) was cast onto the silicon molds using a crosslinker to elastomer ratio of 1 to 5 and cured to create PDMS micropillar arrays.The permeability of each of the three micropillar arrays of depth mentclass2pt{minim29. Time A specified volume of glycerol was injected between the micropillar array and the overlying elastic sheet using a syringe pump. MATLAB was used for image processing to measure the area of the blister and the area of the fluid in the poresThe uncertainty in the time scale  is not large enough to be seen in Fig. SUPPLEMENTARY INFOPeer Review FileSupplementary Movie 1Description of additional supplementary files


Chlorella pyrenoidosa, including herbicides , fungicides (fuberidazole), organic chemical raw materials , disinfectants (trichloroacetonitrile uric acid), and disinfection by-products (trichloroacetonitrile). The results showed that, in addition to specific PSII inhibitors (diuretic and atrazine), other types of pollutants could also quickly affect the photosynthetic system. The photosynthetic fluorescence parameters  could be used to detect the effects of pollutants on the photosynthetic system. Although the decay rate of the photosynthetic fluorescence parameters corresponding to the different pollutants was different, 1 h could be used as an appropriate toxicity exposure time. Moreover, the lowest respondent concentrations of photosynthetic fluorescence parameters to diuron, atrazine, fuberidazole, phenanthrene, P-benzoquinone, phenol, trichloroacetonitrile uric acid, and trichloroacetonitrile were 2 μg·L−1, 5 μg·L−1, 0.05 mg·L−1, 2 μg·L−1, 1.0 mg·L−1, 0.4 g·L−1, 0.1 mg·L−1, and 2.0 mg·L−1, respectively. Finally, diuron, atrazine, fuberidazole, and phenanthrene were selected for a comparison of their photosynthetic inhibition and growth inhibition. The results suggested that photosynthetic inhibition could overcome the time dependence of growth inhibition and shorten the toxic exposure time from more than 24 h to less than 1 h, or even a few minutes, while, the sensitivity of the toxicity test was not weakened. This study indicates that the photosynthetic inhibition method could be used for rapid detection of the toxicity of water pollutants and that algae fluorescence provides convenient access to toxicity data.To achieve rapid and sensitive detection of the toxicity of pollutants in the aquatic environment, a photosynthetic inhibition method with microalgae as the test organism and photosynthetic fluorescence parameters as the test endpoint was proposed. In this study, eight environmental pollutants were selected to act on the tested organism,  Due to the rapid development of industry and agriculture, the types and quantities of pollutants discharged by human beings into the environment are increasing, and the resulting water pollution problems are becoming more and more serious. At the same time, the ecotoxicological data of these pollutants is seriously lacking ,2. Conve2 fixation [2 evolution [The algae growth inhibition method, using biomass as the test index, has been standardized by the Organization for Economic Cooperation and Development  and the fixation , and O2 volution , have beThe fluorescent properties of the chlorophyll of living algae are a powerful tool to evaluate the photosynthetic performance of algae and analyze their protection response ,17. ThroChlorella pyrenoidosa, a common freshwater algae species, which is sensitive to toxicants, fast reproducing, small in size, widely distributed, and easy to obtain, is very suitable for studying the toxicity of environmental pollutants. In this study, C. pyrenoidosa was used as the test organism, and photosynthetic fluorescence parameters were used as the end point of toxicity detection. A series of rapid toxicity detection and quantitative analyses of different water pollutants were carried out under controlled conditions. Furthermore, comparing the results with the traditional growth inhibition method, a fast, sensitive, non-invasive method for detecting and evaluating water toxicity was proposed.Different algae have different sensitivity to toxicants, and there is no possibility of choosing an algae species that is the most sensitive to all toxicants. Based on a consideration of the particular situation, algae species which are easy to cultivate and evenly distributed should be the first choice. In terrestrial freshwater ecosystems, C. pyrenoidosa (FACHB-5) was selected as the test organism and was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences . BG11 medium and 1 L Erlenmeyer flasks for inoculation and culture of C. pyrenoidosa were sterilized by autoclave for 30 min. After aseptic inoculation, Erlenmeyer flask cultures were incubated at 25 ± 1 °C in a constant temperature shaker incubator (MQD-S3R). A white cold-fluorescent tube was used as light source with an automated light/dark cycle of 12 h/12 h, and light intensity 120 μmol (m2·s)−1. All flasks were shaken 2 times daily for 15 min with speed of 120 r·min−1. Before the experiment, the algae were incubated for 3–4 days to reach the exponential growth phase [−1 chlorophyll concentration with BG11 medium for toxicant exposure.th phase . During 680 and photosynthetic fluorescence parameters as the test endpoints showed that DSMO with a volume ratio of less than 2% would not affect the biomass and Photosynthesis of Chlorella pyrenoidosa within 96 h. All treatments were prepared in triplicate.In order to simulate different types of polluted water bodies, we tested 8 common organic pollutants in water, including herbicide , fungicide (fuberidazole), organic chemical raw materials , and disinfectant and its by-products , as shown in −2·s−1. The relative electron transfer rate (rP) was calculated for each layer of photosynthetic light, and at same time a series of photosynthetic parameters were obtained [Photosystem II efficiency was estimated from the photosynthetic fluorescence parameters obtained using a fast repetition rate fluorometer (FRRf) . The fluorometer was equipped with Act2Run system software. An excitation light source was selected according to the absorption spectrum range of different algal species, and a 450-nm LED was selected for green algae. The whole measurement period lasted about 3 min. The samples were exposed to 8 sequential actinic light levels, increasing from 0 to 800 μmol·mobtained ,25.Fv/Fm is the maximum photochemical quantum yield of PSII, which is considered to be related to water splitting and oxygen evolution [F0 is the minimal fluorescence and Fm the maximum level of fluorescence measured after a dark adaptation.volution . The calYield is the effective quantum yield of PSII photochemical energy conversion, which can be calculated as the following equation:Ft and Fm are the steady-state level of fluorescence and the maximum level of fluorescence measured under ambient light, respectively.rP is the relative PSII electron transfer rate, which is derived from the following equation:E is the incident photon irradiance. The fast light curve (FLCs) was constructed with irradiance (E) as abscissa and the relative PSII electron transfer rate (rP) as ordinate, and the initial slope α of the FLCs curve YIE was obtained. α represents the maximum light utilization coefficient, which is obtained by linear regression on the points in the light limited part of the FLC curve.680, as measured on a UV-2550 spectrophotometer  at 680 nm, could be used to quantify the growth of Chlorella [680 was used to represent the biomass of C. pyrenoidosa.Optical density (OD) is an effective indicator to replace the number of algae cells . Previouhlorella . In the p < 0.05). Statistical analyses were carried out using SPSS statistics 22.0.We recorded the values of photosynthetic fluorescence parameters at each exposure time and analyzed the time trend of the parameters. One-way ANOVA was performed on the control group and the treatment group with the addition of different concentrations of toxicants to determine the impact of the toxicants. After one-way ANOVA, the least significant difference method (LSD) was used for post-multiple inspection, to obtain the lowest concentration that had a significant impact on the photosynthetic fluorescence parameters .Although the sensitivity of the four photosynthetic fluorescence parameters  to pollutants was slightly different, their time response trends were consistent, represented by Fv/Fm, as shown in −1 (p < 0.05), 2 μg·L−1 (p < 0.05), 0.05 mg·L−1 (p < 0.05), 2 μg·L−1 (p < 0.05), 1.0 mg·L−1 (p < 0.05), 0.2 g·L−1 (p < 0.05), 0.1 mg·L−1 (p < 0.05), and 2.0 mg·L−1 (p < 0.05), respectively. The results showed that the photosynthetic fluorescence parameters were very sensitive to diuron and atrazine. Unexpectedly, phenanthrene, as a polycyclic aromatic hydrocarbon, was not reported to have a direct effect on the photosynthetic system, but the lowest response concentration of the photosynthetic fluorescence parameters to phenanthrene was very close to that of diuron. Although the above results were only the test results for one kind of algae, it would be facile to include more sensitive or ecologically important species by using the photosynthetic fluorescence parameters of algae for rapid detection of pollutants.One-way ANOVA and post hoc multiple inspection were performed on all photosynthetic fluorescence parameter values after 1 h of exposure. Then we obtained the lowest concentration of pollutants that significantly inhibited the photosynthetic fluorescence parameters in the measured concentration range and took this concentration as the lowest response concentration of the parameters, as shown in The changing trend of inhibition rate of photosynthetic fluorescence parameters  with the concentration of the eight pollutants for the exposure times of 5 min and 1 h are shown in 680). One-way ANOVA and post hoc multiple inspection suggested that diuron, atrazine, fuberidazole, and phenanthrene, within the tested concentration range, began to significantly inhibit the growth of C. pyrenoidosa at 24 h, 24 h, 24 h, and 12 h, respectively (p ≤ 0.05). C. pyrenoidosa exposed to the four environmental pollutants  within 96 h. According to the time effect analysis, the fluctuation of photosynthetic inhibition effect was not very strong, relative to the growth inhibition within the 96 h toxic exposure time, especially with a relatively stable state and a short time (1 h). The growth inhibition was a population-dependent phenomenon, which required time accumulation, and was obviously time-dependent. With the prolongation of exposure time, the growth inhibition rate had an upward trend. Taking diuron (10 μg·L−1) as an example, the relative standard deviation (RSD) of the short-term (5 min−1 h) and long-term (5 min–96 h) inhibition rates of Fv/Fm were 10.4% and 23.1%, respectively. The RSD of the inhibition rate of OD680 (24–96 h) was 32.1%. The process of photosynthesis involves light absorption, electron transfer, photosynthetic phosphorylation, and carbon assimilation. Among them, carbon assimilation is the main source of biomass growth. In theory, the toxicity information obtained through the photosynthetic system was faster and more sensitive. Four pollutants, including diuron, atrazine, fuberidazole, and phenanthrene, were selected to measure the biomass parameter , the lowest EC50-5 min corresponding to diuron, atrazine, fuberidazole, and phenanthrene were 12.74 μg·L−1, 62.94 μg·L−1, 0.55 mg·L−1, and 30.99 μg·L−1, respectively. While, the lowest EC50-1 h corresponding to diuron, atrazine, fuberidazole, and phenanthrene were 9.88 μg·L−1, 62.09 μg·L−1, 0.69 mg·L−1, and 42.37 μg·L−1, respectively. These results indicate that when EC50 was used to evaluate the toxicity of pollutants, and compared with traditional growth inhibition, photosynthetic fluorescence parameters as the endpoint of rapid toxicity detection did not cause an underestimation of toxicity. Compared with Fv/Fm, the toxicity data (EC50) obtained from the photosynthetic fluorescence parameters Yield, α, rP were lower and were more suitable as the end points of the biological toxicity test.Since, photosynthetic inhibition eliminated the dependence on long-term exposure time, we directly compared the results of short-term  photosynthetic inhibition and long-term  growth inhibition, as shown in 680 was used to indicate the growth status of algae cells, and the results show that diuron, atrazine, atrazine, and phenanthrene began to significantly inhibit the growth of C. pyrenoidosa at 24 h, 24 h, 24 h, and 12 h, respectively. Moreover, the corresponding EC50 decreased with the prolongation of the toxicity exposure time. This conclusion was similar to the results of Shi Yajuan et al., in which Chlorella had obvious growth inhibition after 21 h of diuron stress, and the inhibition was significantly enhanced on the 2nd and 3rd days [Scenedesmus obliquus and compared them with growth indicators [Chlorella vulgaris [50 corresponding to 20 min exposure were 72.26 μg·L−1 and 69.02 μg·L−1, respectively. This was similar to the results of this paper, using photosynthetic fluorescence parameters as the end point of the toxicity test, but the measurement parameters of the former study were generally single.The development of fast, simple, affordable, and ecologically-sound toxicity testing methods is very important for improving the supervision of harmful substances and realizing water safety early warning systems. As a test organism, microalgae could obtain many of the related test endpoints faster or easier, such as cell number, chlorophyll content, antioxidant system, oxygen production, and fluorescence emission, etc. Among them, cell number and chlorophyll content are closely related to indicators of algae growth and biomass, so the recording of toxic effects requires time to accumulate ,30. In t3rd days . Changes3rd days . Jiang Ldicators . The resvulgaris . The resIsochrysis globosa within 72 h [Euglena agilis Carter by Kottuparambil S et al. demonstrated that phenol significantly reduced the maximum quantum yield (Fv/Fm) and maximum photosynthetic electron transport rate (rPmax) of the photosystem (PSII) within 1 h [50 obtained by Fv/Fm was higher than that obtained by OD680. Moreover, the dose–effect results of phenanthrene and diuron show that the EC50 obtained from Fv/Fm was approximately twice that of α, rP, and Yield. Therefore, more consideration should be given to the application of the parameters α, rP, and Yield in toxicity testing.The fluorescence kinetics method applied to the measurement of phytoplankton aquatic toxicity has the characteristics of a simple measurement process, no sample pretreatment, and a non-invasive measurement process. Most of the previous related research focused on a single toxicant, and the time of exposure to the toxicants ranged from a few hours to a few days. Pérez et al. studied the effects of polycyclic aromatic hydrocarbons on the photosynthetic fluorescence parameters of hin 72 h . Tiam ethin 72 h . The acuthin 1 h . Howeverthin 1 h ,37. HowePhotosynthesis is the most important physiological process in phytoplankton and is the basis of its growth. Any inhibition of its growth should be preferentially reflected in the photosynthetic process. Rapid detection of eight common pollutants showed that the inhibitory effect of the pollutants could be reflected in the photosynthetic fluorescence parameters  within a few minutes. Moreover, the method of photosynthetic inhibition was not unique to direct PSII inhibitors (diuron and atrazine), and it could also achieve rapid toxicity detection for pollutants not directly acting on the photosystem II. Diuron, atrazine, fuberidazole, and phenanthrene were selected for a comparison of photosynthetic inhibition and growth inhibition. Compared with the 3–4 day growth inhibition method, the photosynthetic inhibition method had a considerable time saving advantage, which provided a new method for the emergency monitoring of water pollution accidents and the rapid assessment of ecological environment risk. A bioassay method based on the rapid changes of photosynthetic fluorescence parameters has the most potential for field testing. This study did not involve the study of algae species differences. However, in the future, a variety of test objects could be customized according to the ecosystem or interested groups, to study the impact of toxic pollutants on a set of algae species, so as to provide a more ecologically representative response.


It has been known in the industrial sector that in closely intermeshing counterrotating twin screw extruders, large separating forces develop in the calender gap, which push the screws towards the barrel wall. The result is significant wear in the region defined by 30°- and 60°-degree angles from the vertical. In the present investigation, pressures were measured around the barrel in extrusion of two rigid PVC resins in a laboratory extruder of 55 mm diameter and the forces on the screw core were determined. Numerical flow simulations were also carried out using the power-law viscosity parameters of the resins. From the experimental results, it was determined that the resultant forces are in the 30 degree angle direction, and from the computer simulations, the angle is between 18° and 25°. It is argued that the resultant force angle will be somewhat larger in large diameter extruders, due to the additional contribution of gravity. The counter-rotating intermeshing twin screw extruders are used widely for pipes, profiles and sheet extrusions. The main advantage over single screw extruders and co-rotating twin screw extruders is their positive displacement pumping, which imparts little frictional heating to temperature sensitive materials, such as PVC. Of course, positive displacement implies limited mixing. The screws form helically distorted C-shaped chambers, as explained in various books ,4,5,6,7,Twin-Screw Extruders, that circumstances and politics played at least some role in classical co-rotators receiving 20 to 50 times greater investments of time and money than classical counter-rotation”. Thiele goes on to acknowledge the limitations in mixing in counterrotating twin screw extruders (TSEs) and to state that: “At high screw speeds the calender pressures became so great as to cause the melt film against barrels opposite the intermesh to fail; that is, the extruder could eat itself. For that reason, production classical counter-rotators were generally limited to 150 RPM and below”. In fact, large diameter closely intermeshing counter-rotating TSEs are limited to less than 50 RPM, according to Martin [Historically, counter-rotating twin screw extruders have received significantly less research and development efforts than corotating. According to Thiele  “Dr. Whio Martin . For larIntermeshing counter-rotating screws usually rotate outward. It is known in the industrial sector and it has been reported in some books and publications ,15,16,17A parallel closely intermeshing counter-rotating twin screw extruder of 55 mm diameter and L/D ratio of 20 was used in the experiments. The metering zone has a length of 300 mm and double flight screw geometry with screw pitch of 50 mm, core diameter of 31 mm, screw-barrel gap 0.1 mm, calender gap 0.55 mm and screw flank gap 1.44 mm, as shown in m = 89,120 Pa∙sn and n = 0.434 at 190 °C, and for resin B, m = 149,818 Pa∙sn and n = 0.4 at 190 °C. Two different uPVC powder grade formulations , provideBy using the adjustable valve in the die, it was possible to generate high pressures in the metering zone and determine to what extent the extruder was filled with molten material. This could be observed from the pressures recorded along the barrel P1, P2, P3, P4, P5 and P6. At location 5, pressures were measured around the barrel at the positions P5.1, P5.2, P5.3 and P5.4, as shown in m is the consistency index, n the power-law index, U the rotational speed, Ho the minimum gap, R the radius of roll and n) is a function of n equal to 0.75 for n = 0.434 and equal to 0.78 for n = 0.4.It can be seen that the pressures measured increase, from the 60° to the 300° position as the molten PVC is dragged to the nip region. The differences between the maximum (at 300°) and minimum (at 60°) recorded pressures vary from 4.7 MPa to 6.4 MPa for resin A and 5.8 MPa to 7.1 MPa for resin B. The differences are somewhat higher at higher back (adapter) pressures, because the viscosity of polymers increases with pressure . From thR = (55 + 31)/2 = 43 mm, for resin A we obtain Pmax = 3.96 MPa and for resin B Pmax = 5.99 MPa. These values are close to the pressure differences measured, despite the fact that Equation (2) was derived for two equal diameter rolls, from an analysis of flow in the vicinity of the minimum gap, and there is no barrel wall involved in the calendering process. Consequently, a rough estimate of the separating force can also be obtained, from Middleman [L is the length of the roll (core of the screw) and n) = 0.3 for n = 0.434 and F(n) = 3.2 for n = 0.4. This equation gives F/L = 38,104 N/meter for resin A and F/L = 59784 N/meter for resin B. Assuming that the force is exerted on the entire length of the screw in the 30 cm metering zone, we have for resin A, F = 11,431 N (1.16 ton-force) and for resin B, F = 17,935 (1.83 ton-force). Obviously, under the action of a separating force of over one metric ton, the screws will be pushed towards the barrel wall and exert abrasive action, as explained also in the introduction of this paper.From Equation (2), assuming 2iddleman :(3)FL=mUx and the y directions can be made from the measured pressures at positions P5.1, P5.2, P5.3 and P5.4. These pressures were plotted in S = Rdθ will be p(θ)cosθdS = p(θ)cosθRdθ and −p(θ)sinθdS = −p(θ)cosθRdθ, respectively. The forces per unit length can be obtained by numerically integrating the pressures shown in Another calculation of the separating forces both in the y axis.These two equations are used routinely for load determination in the lubrication of journal bearings ,20,21. TRe << 1) and isothermal. Under these approximations the Navier-Stokes equations are simplified to:p is the pressure and τ is the stress tensor. The Generalized Newtonian fluid is used to relate the fluid stresses with the rate of strain given by:η is the non-Newtonian viscosity of the material and D is the rate of strain tensor given by:U is the velocity vector. We further assume that the fluid rheology is described by the power-law model:m is usually referred to as the consistency index (Pa·sn), n is the power-law index (for n = 1 Newtonian fluid) and IID is the second invariant [D. It can easily be shown that this generalization reduces to the power-law fluid model for simple shear flow, i.e., Equation (1).Various aspects of flow analysis and computer simulations of counter-rotating TSEs ,29,30,31given by:τ=η2DwheM = 3 × 104. This mesh is chosen after a mesh-independence study. We have constructed a coarse mesh of cM = 104 volumes and a dense mesh with Md = 9 × 104 elements. Subsequently, we compared the values of the maximum velocity and the pressure at the center of the minimum calender gap. We found that these values changed by less than 0.1% from mesh M to Mc. For boundary conditions, we assumed the no-slip condition at the surface of the barrel and the surface of the screws. The governing equations are solved iteratively using the SIMPLE [−7.For the discretization of the computational domain, we use a combination of regular and unstructured meshes consisting of triangular volumes. All meshes are constructed with the GMSH software . The unse SIMPLE  pressuree SIMPLE . For theIn The problem of pressure peaks and valleys canceling each other has been studied extensively ,43,44 inThe determination of the resultant forces on the screw core has been accomplished by integrating the local pressure forces around the circumference. The experimental measurements of pressure were made at 60°, 120°, 240° and 300° clockwise from the minimum gap and the values were fitted to straight lines. It was not possible to make measurements just upstream of the nip, where the pressure was expected to attain the highest value. It was decided to assume that the maximum pressure is reasonably approximated by extrapolations of the straight lines to 360°. The integrations gave resultant force angles of about 30° clockwise from the vertical. For the computer simulation, a two-dimensional flow analysis was carried out on a plane normal to the screw axis. As expected, negative pressures were obtained in the divergent section, which cancel the positive pressures in the convergent section. Using boundary conditions similar to those used in journal lubrication analyses, the negative pressures were removed and the subsequent integrations gave resultant force angles between 18° and 25°.In the industrial sector, it is frequently said that the screws move to directions popularly referred to as the ten o’clock and two o’clock positions, which correspond to 60° degrees from the vertical. Diameter measurements of the 90 mm extruder shown in In the present investigation (55 mm diameter extruder), the effect of gravity on the screw was not taken into account, because it is relatively small when compared to the separating forces. However, in large diameter extruders, a screw might be four meters long having a weight of a couple of tons. Such screws will be subjected to bending like cantilevered beams. The resultant force angle of calender separating force and gravity will be somewhat larger than those calculated in the present investigation.


Rangifer tarandus, the northern species including both reindeer and caribou, is a pillar of northern ecosystems and the lives of northern peoples. As the only domestic cervid, reindeer are important not only to the herders and hunters who presently interact with them, but also to zooarchaeologists and palaeontologists tracing their histories. Unfortunately, limited anatomical information on Rangifer tarandus muscles is available beyond descriptions of the large muscle groups. The lower limb and hoof in particular is poorly documented. This is problematic, as this important body part has the potential to be informative in zooarchaeological analyses of habitual activity, especially in regards to historical animal health, movement, and habitual activity. Better understanding of the hoof can additionally be useful to herders and veterinarians seeking to provide veterinary care for living animals. This study has used dissections and comparisons of the reindeer hoof with other domestic ungulates to document both the common and unique structures in Rangifer tarandus hooves, including the presence and attachment points of these structures. As these structures have proved unique, especially in regards to the dewclaw, it is important that other ungulates not be used exclusively in the analysis of Rangifer tarandus remains. Rangifer tarandus anatomy persist. This is particularly true when discussing the hooves of reindeer. Veterinarians, hunters, and herders have first-hand knowledge of the anatomy and physiology of the reindeer hoof, but no guide for the novice has yet been produced. This study presents the origins, insertions, and primary actions of the tendons and ligaments of the hoof in order to further knowledge of Rangifer biology and anatomy.While reindeer and caribou are and have been a keystone species for human survival and ecological stability in the Arctic and sub-Arctic north, gaps in our knowledge of In human osteology, osseous changes at the points of muscle, tendon, or ligament insertion, known as entheseal changes, are commonly examined to help reconstruct habitual activity  and metatarsal (MT) bones are similar in shape, with a few notable differences Figs. , 3. BothThe round, spool-shaped trochanters on the head of the metapodials provide the platform for articulation of the first phalanx (PI) and the proximal sesamoid. The cup-shaped concavities on the sides of the metapodial heads provide the origin for the abaxial metacarpo- and metatarso-phalangeal collateral ligaments. The deep indentation between the trochanters of the metapodial head are alternately referred to as the intercapital or intertrochanteric groove, which serves as the origin site of the axial metacarpo-/metatarsophalangeal collateral ligaments.The fetlock joint is located where the metapodial articulates with the first phalanx, as well as with the proximal and dorsomedial sesamoids. The metacarpophalangeal joint is held together with collateral ligaments on the abaxial surfaces, axial collateral ligaments between the metacarpal and the first phalanx, and smaller cruciate ligaments supporting the plantar  sesamoids of each digit. The collateral ligaments of the fetlock joint run from the cup-shaped concavities on the abaxial aspects of the metacarpal capita and then divide, with one ligament going to the proximal plantar surfaces of the first phalanges. The other ligament combines with the cruciate ligaments holding the proximal sesamoids to the first phalanx. The large axial collateral ligaments, which attach the metacarpal to the first phalanges, run from each side of the interior intercapital notch of the metacarpal capita to combine with the axial portion of the small cruciate ligament of the same digit. These small cruciate ligaments, along with small sesamoidean ligaments, attach the metacarpal and proximal sesamoids to the axial eminences along the proximal axial and palmar surfaces of the first phalanx. The fibers of these cruciate ligaments run perpendicularly to the shaft of the first phalanx and may be distinguished by the direction of osseous build-up at the attachment point. This site is adjacent to the attachment point for the proximal annular ligaments, whose attachment site runs along the plantar surface, rather than extending along the axial surface.manicae flexoriae) around the deep flexor tendon until just proximal to the pastern joint. The combined tendons bifurcate at the fetlock joint to follow the individual digits III and IV. The interosseous ligaments run deep to the flexors. The interosseous ligaments begin as a thin, flat, fibrous structure that lays flat against the metacarpal. At the fetlock joint of the forelimb, the interosseous ligament divides into seven branches. The axial and abaxial extensor branches (divided into interosseous III and IV dependant on the digit which they serve) run on either side of each digit, while the medial branch inserts into the intercapital notch of the metapodial. The axial branches run on the internal surface of the hoof, crossing from the plantar to dorsal aspect of the hoof. The abaxial branches follow an analogous path on the outer surface of the hoof. The abaxial branch also subdivides, providing a small tendon to the proximal sesamoid bone. The axial and abaxial extensor branches of the interosseous ligaments recombine on the dorsal surface of the hoof, joining with the medial and lateral extensors at the extensor junction of the pastern joint. Two small accessory ligaments also subdivide, with the interosseous accessory ligament for paradigitii V subdividing from interosseous IV and the accessory ligament for paradigitii II subdividing from interosseous III. These ligaments attach to the axial aspect of the stylets of the dewclaws in the forelimb before continuing to a second attachment site on the proximal abaxial surface of PI proper and act to stabilize the dewclaw.Two layers of tendons run down the dorsal side of the metacarpal Fig. . SuperfiThe proximal sesamoids of the hoof sit against the base of the first phalanx and provide a larger articular surface against which the rounded head of the metacarpal hinges. The entire fetlock joint is encased in a thick band of fascia covering the network of suspensory ligaments, as well as the tendons which pass along the dorsal and plantar sides of this joint. Superficially, the palmar annular ligament of digital flexors holds the tendon of the superficial and deep digital flexors in place, while the thin abaxial extensor branches run deep to this, further supporting the proximal sesamoids. They are held in place by the digital flexors until diverging just distally to the fetlock joint, where they curl around the axial and abaxial surfaces of the first phalanx before joining the other extensor tendons on the dorsal aspect of the digit.The dorsal compartment of this joint contains far less soft tissue than the plantar, as the belly of the combined superficial and digital flexor tendons are by far the most robust soft structure of the hoof. Along the dorsal portion of the joint there run two layers of extensor tendons, all contained within a single tendon sheath. The common digital extensor tendon is a thin, flat band which runs superficially along the dorsal surface of the metapodial, bifurcating at the fetlock joint into the digital extensor tendons for digits III and IV. Deep to this, the lateral and medial digital extensors run, crossing the joint and housing, in the forelimb, the dorsomedial sesamoid bones, one on each digit. These small bones act as miniature patellae, facilitating the extension of the first phalanx at the metacarpophalangeal joint Hull . It is sRangifer tarandus hooves wildly deviate from those of Bos taurus. While the tendons and ligaments mentioned above occur in different configurations and slightly different sites, they are nonetheless identifiable as analogous structures. Generally speaking, there are both more in number and more complex suspensory ligaments in the fetlock joint of Rangifer tarandus than in Bos taurus. These provide flexibility, especially in the abduction of the digits, at the cost of greater stability provided by the thick plantar annular ligament in Bos taurus. This is expected considering differing habitats and behaviors. The Rangifer tarandus dewclaw, however, has structures without analog in Bos taurus, which are described below  also run from the proximal and distal ends of the dewclaw first phalanx. The proximal accessory extensor ligament combines with the abaxial extensor branches, and with the medial and lateral digital extensors, insert at the proximal dorsal aspect of the second phalanx of the digits proper. The action of these ligaments causes the abduction of the dewclaws to extend the digits of the hoof, causing the hoof to splay out, increasing the surface area. The additional distal accessory ligament runs between the distal axial surface of the dewclaw to the proximal abaxial surface of the third phalanx proper, allowing the dewclaws to flex in concert with the other digits of the hoof. They are unable to flex independently. On the forelimb dewclaw, additional small interdigital collateral ligaments run between the small first and second phalanx, mirroring those of the proximal collateral interdigital ligaments on the hoof proper.Several structures attach to the first phalanx between the fetlock and pastern joints. On the proximal dorsal aspect of the first phalanx, one insertion site of the deep medial and lateral extensor tendons occurs, keeping the dorsomedial sesamoid in place. On the plantar aspect, two important attachment sites produce two bilateral ridges along the shaft of the bone. These ridges are the attachment points for the proximal and dorsal digital annular ligaments, which encircle the strong flexor tendons of the digits. These ligaments are the primary stress points on the first phalanx when the digits are flexed.The metatarsophalangeal joint is similar to the metacarpophalangeal joint in structure. The collateral and sesamoidal ligaments of the metatarsal follow the same pattern as those of the metacarpal, as do the superficial and deep flexors. The interosseous accessory paradigital ligaments, however, are much more gracile and insert on PI of the dewclaw, as no stylets are present in the hind limb. In the dorsal compartment, the extensors follow a similar pattern, with the long digital extensor running superficially to the medial and lateral extensors, following a path analogous to the common digital extensor of the forelimb.The tendons of the forelimb dewclaws are more developed than those of the hindlimb, and the forelimb dewclaws are both more robust and contain an additional bone: the vestigial MC II/V, or stylets. Despite this, they retain the same general tendinous and ligamentary structures, with a few important exceptions. Proximally, the dewclaws are held in place by fascia and the digital accessory branch of interosseous III/IV in the forelimb. This ligament runs the length of the stylet and inserts on the first phalanx of the forelimb dewclaw and continues to insert on PI of the digit proper. In the hindlimb, this tendon attaches to and terminates at the first phalanx of the dewclaw. Because of the lack of stylet and reduced size of the first phalanx of the hindlimb dewclaw, the dewclaws are not able to abduct as broadly as in the forelimb. In the forelimb, the proximal accessory ligament of paradigitii II/V runs from the axial surface of the stylet of MC II/V, while in the hindlimb it originates from the proximal aspect of the first phalanx of MT II/V to attach to the extensor junction on PII of the digits proper. The distal accessory ligament of paradigitii II/V remains analogous. While small interdigital collateral ligaments are present on the hindlimb dewclaw, they are less developed than those of the forelimb, and the dewclaw itself has less flexibility between the joints of the dewclaw phalanges.The proximal interphalangeal or pastern joint between the first and second phalanges is less complex than that of the fetlock joint Figs. , 5. Two The medial and lateral digital extensors, axial and abaxial extensor branches of the interosseous ligament, and the proximal accessory paradigital ligament of the dewclaw insert on the dorsal surface of the body of the second phalanx at the extensor junction. This is a diffuse insertion site, much less discreet than that of the flexor tuberosity on the plantar side, where the superficial flexor tendon inserts, and the insertion site forms a noticeable trapezoidal platform. The medial and lateral digital extensor tendons bifurcate, with one branch inserting along the distal dorsal aspect of the second phalanx, and the other branch continuing to the third phalanx. The abaxial and axial extensor branches likewise partially insert at the extensor junction, then divide again, and continue to the third phalanx. Just below the pastern joint is a large interdigital ligament which is cruciate in form, which extends across the entire axial surface of the second phalanx and inserts at and just distal to the coffin joint on the second and third phalanges. This large, strong structure is without elasticity, providing protection against over-abduction of the hoof.Odocoileus virginianus. This interdigital gland secretes kairomones to parasites and antimicrobial substances in O. virginianus .Between the pastern joint and the distal cruciate interdigital ligament a large exocrine gland is housed, present in both the fore- and hindlimb. This gland is also present in other cervids, notably s Hewitt . Reindees Hewitt . It may The distal interphalangeal joint, or coffin joint, is covered by the transition from the dermis to the cartilaginous hoof capsule Fig. . This joThe abaxial digital extensor and the proximal accessory digital extensor of the dewclaw partially combine with the other digital extensors at the pastern joint, but both also have branches running to the third phalanx. Both curl around the second phalanx after partially inserting on the dorsal surface, terminating on the plantar articular border of the third phalanx. Finally, the distal accessory paradigital ligament from the dewclaw extends to insert at the parietal groove on the abaxial surface of the third phalanx.The long, thin, fibrous common digital extensor tendon of the forelimb and the long digital extensor tendon of the hindlimb insert on the extensor tubercle, an ovoid osseous structure at the proximal dorsal axial part of the third phalanx. Abaxial to this, along the border of the articular surface of the dorsal axial aspect of the third phalanx, the long branches of the medial and lateral digital extensor tendons insert on a small ridge of bone on the proximal edge of the abaxial surface. A small sesamoidal ligament runs from the proximal aspect distal sesamoid to the third phalanx, while the impar ligament runs from the distal aspect of the distal sesamoid to the third phalanx, further securing it in place. The deep digital flexor, after partially inserting at the flexor tubercle and passing over the distal sesamoid, fans out to make a diffuse insertion across nearly the entirety of the plantar surface of the third phalanx.Rangifer tarandus, which then gives way to the coronary segment, extending down the rest of the dorsal portion of the hoof. The sole segment covers the cranial point of the hoof, while the bulbar segment covers most of the plantar surface of the hoof and covers the hoof bulb, internally. The hoof bulb is a highly vascularized cushion of soft tissue covering the caudal portion of the internal hoof, running superficially to the deep digital flexor. This hoof bulb is innervated and vascularized by nerves, veins, and arteries running the length of the metapodials and digits to branch out into a network in the hoof capsule, with veins and arteries running through the abaxial and axial foramina of the third phalanx.Compared to the rest of the metapodial and digits, the hoof capsule is much more densely packed with vascularized soft tissue structures. The surface of the hoof is divided into several parts. As per the definitions given in  in Bos taurus is represented by the stifle, which descends from the proximal end of the metapodial, while the bones of the dewclaw are represented by one or two tiny bones (Budras and Habel Rangifer tarandus is highly functional, especially in the forelimb, where the vestigial MC II/V form stylets which are connected by branches of tendons and activate in concert with the digits proper. The bones of the dewclaw in both fore- and hindlimb have identifiable first, second, and third phalanges. In addition, the strong accessory abaxial extensor tendon branches of the fore- and hindlimb dewclaw act to stabilize the hoof and prevent over-abduction. This creates a much larger hoof surface for locomotion and foraging. This unique adaptation shows the efficiency of the Rangifer tarandus body in snowy environments, but this may also open the hoof up to different injury and stress patterns compared to feet of other ungulates, a subject which has been explored in other species in zooarchaeology (Izeta gy Izeta .Rangifer tarandus such as R.t. caribou and R.t. granti in North America and high Arctic ecotypes of the far North such as R.t. platyrhynchus would further expand our understanding of Rangifer tarandus anatomy. While this study provides only the basic anatomy of Rangifer tarandus hooves, it is hoped that this research will lead to more understanding of reindeer and caribou anatomy and assist in answering questions in both the past and present.This study is limited, as it was done only on the limbs of a small number of domestic reindeer, and therefore may not represent the full range of variation present in the global populations of this species. Further research into the hoof anatomy that includes other subspecies of