We found that a subset of vCA1 neurons were responsive to the aversive shock during context conditioning, their activity was necessary for memory encoding, and these shock-responsive neurons were enriched in the vCA1 projection to the amygdala. During memory retrieval, a population of vCA1 neurons became correlated with shock-encoding neurons, and the magnitude of synchronized activity within this population was proportional to memory strength. The emergence of these correlated networks was disrupted by inhibiting vCA1 shock responses during memory encoding. Thus, our findings suggest that networks of cells that become correlated with shock-responsive neurons in vCA1 are essential components of contextual fear memory ensembles.Ventral hippocampal CA1 (vCA1) projections to the amygdala are necessary for contextual fear memory. Here we used in vivo Ca The vCA1-BA projection is enriched in shock responsive neurons, which are necessary for fear memory encoding and become correlated with a network of neurons during retrieval. Here the authors show that the magnitude of vCA1 correlated activity is proportional to memory strength and requires the shock response during encoding. While the HPC has classically been considered a purely cognitive structure that processes contextual and spatial information independent of valence, a growing body of work has also supported a role for the HPC in emotional behavior14. This dual function is thought to be segregated along the dorso-ventral axis of the HPC, with the dorsal HPC involved in cognitive processes, and the ventral HPC (vHPC) involved in emotional behaviors16. The specialization of the vHPC in emotional behavior is further supported by recent work which identified vHPC representations of aversive stimuli that are necessary for anxiety-related behavior12.The hippocampus (HPC) is critical for the formation of contextual memories24. One such functional distinction has recently been identified between the projections to the lateral hypothalamus (LHA) and basal amygdala (BA), wherein ventral hippocampal CA1 (vCA1) projections to the BA, but not LHA, are necessary for contextual fear conditioning9. Still, how representations of aversive stimuli may be incorporated into emotional memories within the vHPC, and the neural circuit mechanisms by which vCA1 projections to the amygdala mediate contextual fear memory formation and recall remain unknown.Within the vHPC, functional specialization is delineated into distinct subsets of neurons that project to specific limbic structures to mediate unique aspects of behavior9, we first assessed the activity patterns in these two cell populations to identify signatures of activity during contextual fear conditioning that are specialized to the vCA1-BA projection. We utilized a retrograde CAV2-Cre virus in the BA or LHA to selectively express Cre-dependent GCaMP6f in these projections as previously described9, which allowed us to record and track the activity of sparse populations of vCA1-BA and vCA1-LHA projecting neurons  recapitulated the effects observed at the R ≥ 0.3 significance threshold . We found that across all metrics, the increase in correlated activity of vCA1 shock cells and their A2 partners was positively correlated with the strength of memory retrieval across animals Fig. . These rConsidering that the strength of memory retrieval was proportional to the magnitude of correlation change within the network of vCA1 shock cells and their A2 partners, we next assessed whether the vCA1 shock response in context encoding was necessary for the formation of a contextual fear memory. To assess this, we virally expressed a Cre-dependent red-shifted opsin Chrimson or mCherry no-opsin control virus within vCA1 of vGAT-Cre mice bilaterally and implanted fiber optics to deliver laser stimulation during behavior Fig. . We then2+ imaging and unilateral optogenetic silencing manipulation to selectively inhibit vCA1 activity during the shock period in context encoding, and assessed the effect on correlated activity during retrieval  during retrieval.We next investigated whether the vCA1 shock response during context encoding was necessary for the emergence of correlated activity during retrieval, or if these correlated networks would form independently of aversive shock representations at the level of vCA1. To accomplish this, we employed a simultaneous Caval Fig. . Additiolls Fig. . We founlls Fig. . We thenlls Fig. . As expells Fig. . In addi A2 Fig. . Howeverice Fig. . This efice Fig. . Moreoveice Fig. . Thus, t6, the dynamic signatures of cell activity by which the HPC encodes and retrieves these contextual memories is not well understood. Both experimental data35 and computational models38 have proposed that synchronous activity within the HPC may facilitate long-term synaptic plasticity for memory formation and retrieval. Our work identifies a putative signature of memory retrieval within synchronous ensembles of neurons in vCA1 which is related to memory strength, and incorporates neurons responding to the unconditioned stimulus during training. Moreover, we demonstrate that the vCA1 shock response during context encoding is necessary for the emergence of these functional correlation networks during memory retrieval. This finding may be explained by recent studies which have found that increased neuronal excitability promotes cell recruitment into a memory trace or engram39, as neuronal excitability enhances synaptic plasticity and promotes synaptic strengthening38. Therefore, by extension, the shock may result in increased recruitment of vCA1-BA projecting neurons (as this projection is enriched in shock-responsive neurons) to the contextual fear memory engram by increasing neuronal excitability in vCA1 at the time of associative memory encoding. Importantly, the correlations described here are captured over slow time scales (within 1 s time bins), which may be mediated by subthreshold activity or Ca2+ plateau potentials which have recently been described in a CA1 synaptic plasticity rule which operates over the timescale of seconds40. Finally, when considering why correlation with vCA1 shock-responsive neurons may promote memory retrieval, we propose two possible mechanisms. First, considering that only ~50% of vCA1-BA projecting neurons are responsive to aversive shocks, vCA1 shock cells that project to the amygdala may form a functional correlation network with other vCA1-BA projecting neurons that are not responsive to the aversive US, but rather encode a representation of the conditioned context. In this model, correlated activity between these projecting neurons could lead to enhanced communication between the HPC-amygdala during contextual fear memory retrieval by increasing synchronized synaptic release within the BA, which would then engage downstream circuits to promote freezing behavior  will therefore be critical for our ability to understand and improve long-term memory.While decades of work have established a role for the HPC in contextual memory based on static markers of prior cell activityior Fig. . Alternaior Fig. . This coior Fig. , whose i42 were bred on a C57BL/6J background. Mice were used for experiments at 8 weeks of age and provided with unrestricted access to food and water. All mice were housed 2–5 per cage on a 12 h light/dark schedule with lights off at 6:00 p.m., with ambient temperature ~74 °F and ~20% humidity. Experiments were conducted during the light portion.Procedures were conducted in accordance with the U.S. NIH Guide for the Care and Use of Laboratory Animals and the New York State Psychiatric Institute Institutional Animal Care and Use Committees at Columbia University. Adult male C57BL/6J mice were supplied by the Jackson Laboratory, and vGAT-IRES-Cre mice12 vg/ml . For calcium imaging, viruses  were packaged and supplied by UPenn Vector Core at titers ~6 × 1012 vg/ml and viral aliquots were diluted prior to use with artificial cortex buffer to ~2 × 1012 vg/ml. The CAV2-Cre virus was packaged and supplied by the Larry S Zweifel laboratory in collaboration.Adeno-associated viruses for optogenetic manipulations were packaged and supplied by the UNC Vector Core Facility at titers of ~4–8 × 102) and head-fixed in a stereotax . Ophthalmic ointment was applied for eye lubrication, fur was shaved and incision site sterilized, and body temperature maintained with a T/pump warm water recirculator . Subcutaneous saline and carpofen were provided peri-operatively and post-operatively for 3 additional days for analgesia and hydration.For all surgical procedures, mice were anesthetized  were inserted around the implantation site, a craniotomy was made using a dental drill and dura removed from the brain surface with fine tweezers and a stream of sterile saline and absorptive spears (Fine Science Tools (FST), Foster City, CA). Mice were then injected unilaterally  with 500 nl of GCaMP6f with a Nanoject syringe  followed by implantation of a ~0.5 mm diameter, ~6.1 mm long GRIN lens  over the injection site. The lens was slowly lowered in 0.1 mm DV steps to the target depth  and then fixed to the skull with dental cement . At the completion of surgery, the lens was protected with a cap of lens paper and liquid mold rubber , and imaging experiments were started 3 weeks later. For CAV-Cre retrograde surgeries, CAV-Cre virus was injected at the following coordinates ; For vGAT-Cre surgeries (on a pure 129 background strain) the following ML coordinates were used in order to target vCA1: viral injection site ML 3.75 mm, lens implantation site ML 3.90 mm). For simultaneous vCA1 optogenetic and Ca2+ imaging, GCaMP6f and opsin virus were pre-mixed  prior to injection into brain tissue.For in vivo CaFor bilateral optogenetic silencing experiments , undiluted opsin virus was injected at the following coordinates  and fiber optics were implanted at the following coordinates .Context A: anise scent, white noise, floor shock bars exposed, shock box doors closed, and a light on within the chamber, and were allowed to explore the context for 3 min prior to receiving a 2 s 0.7 mA strength foot shock. On day 2, mice were placed back into the same context for 3 min to assess for freezing during CFC retrieval. On day 3, mice were placed in a modified fear conditioning box for 3 min in the same room but with the following modified contextual cues: Context B: lemon scent, no white noise, floor shock bars covered with an insert and with bedding, shock box doors open so that mice could see distal cues within the behavior room, and chamber light off. For tone–shock conditioning, at the end of the 3 min of day 2 retrieval in Context A, mice were treated with 3 tone–shock pairings, with a 2 s foot shock delivered in the last 2 s of a 20 s, 2 kHz, 90 db tone. On day 3 in Context B after 3 min of novel context exploration, the 20 s tones were presented to mice to assess for tone-retrieval . Behavior was recorded with EthovisionXT 11 video software, and freezing was hand-scored by a blinded experimenter using ObserverXT 12 scoring software .For CFC imaging, mice were run through a 3-day contextual fear conditioning paradigm. On day 1, mice were placed in a standard fear conditioning shock box  with the following contextual cues: 43. The protective rubber mold was removed from the lens and lens cleaned with ethanol wipes and lens paper. A magnetic baseplate was screwed onto the miniscope and lowered over the implanted GRIN lens until the FOV came into focus. The baseplate was dental cemented in place onto the mouse headcap if GCaMP+ neurons were visualized. After drying the baseplate was unscrewed from the miniscope and miniscope was removed from the animal’s headcap, and baseplate was covered with a customized lens cover . Once baseplated, the same microscope was used for every imaging session with that mouse and the focal plane of the microscope was left unchanged in order to ensure a constant FOV across imaging sessions. Imaging sessions were started the following day. Prior to each imaging session, mice were briefly anesthetized (<5 min) in order to attach the miniscope to the baseplate, and a snap shot of the FOV was taken and matched between days to ensure exact placement of the miniscope within the baseplate in order to maintain a constant FOV across days. All mice were allowed to recover from anesthesia for 30 min before beginning imaging, and Ca2+ videos were recorded with nVista and nVoke acquisition software , and behavior and Ca2+-imaging video acquisition were synchronized with a TTL-triggering system (a TTL pulse from EthoVision XT 10 and Noldus IO box system was received by nVista and nVoke DAQ boxes to commence imaging sessions). Ca2+ videos were acquired at 15 or 20 frames per second with 66.56 ms exposure, and the same LED power settings were used for each mouse throughout the series of imaging sessions.Three weeks after surgery, mice were head-fixed on a stereotactic frame while under 1.5% isoflurane with 1 L/min oxygen flow in order to check GRIN lenses for GCaMP expression with a miniaturized microscope 10. EthoVision XT 11 software and a Noldus IO box system were used to record live-tracking of mice while they explored the CFC box and to trigger the laser ON during the specified time period.Four-week after viral injection and fiber-optic implantation, mice were handled and habituated to fiber-optic adapter cables for 2 days prior to beginning behavioral experiments. For vGAT-Chrimson stimulation, ~5–8 M were delivered via a 523 nm 100 mW laser  to fiber optics implanted in mouse brain using a fiber-optic patch cableSimultaneous calcium imaging and optogenetic manipulation was performed using the nVoke miniature microendoscope from Inscopix. Briefly, the nVoke system uses two LEDs  with different wavelengths to simultaneously excite GCaMP for imaging and activate a red-shifted opsin (in this case Chrimson-mCherry) for optogenetic manipulation. Custom code written through Med-Associates software was used to control the fear conditioning boxes and TTL connection with the Inscopix system allowing for precise timing of LED and shock delivery.For nVoke imaging, a 3-day CFC paradigm was performed as described above. On the first day of CFC, during a period extending from 2 s before the shock to 2 s after the shock  continuous illumination with the 590 nm LED was delivered unilaterally to vCA1 through the implanted GRIN lens. On days 2 and 3 of the CFC paradigm no amber LED stimulation was given. Immediately after the 3rd day of CFC mice were returned to their homecages and received alternating ON–OFF epochs of amber LED stimulation while simultaneously imaging GCaMP activity. Six epochs (each 30 s) of amber LED stimulation were interspersed with seven epochs (each 60 s) without amber LED stimulation.For histology, mice were perfused transcardially with 4% paraformaldehyde (PFA) in 1X phosphate buffer solution (PBS), after which brains were removed and post-fixed in 4% PFA for 24 h. They were transferred to a 30% sucrose solution in 1X PBS for 2 days, after which they were flash-frozen in methylbutane and coronally sliced on a cryostat (Leica CM 3050S) at a thickness of 50 µm. Prior to mounting brain sections and cover slipping with ProLong Gold antifade reagent , they were incubated with 1:100 Hoechst  in 1X PBS for 10 min to label cell nuclei. No immunolabeling was applied to visualize fluorophores, as endogenous viral expression was sufficiently bright in histology preparations. After mounting, brain sections were imaged on a (Leica TCS SP8) confocal microscope using a ×10 or ×20 objective, or a (Zeiss Axiovert 200) epifluorescent microscope using a ×2.5 or ×10 objective.In order to determine appropriate GRIN lens placements after imaging, brains were post-fixed with head-caps, lenses, and skulls in place for 1 week in 4% PFA to harden the GRIN lens tract within the brain tissue for better visualization. Brains were then transferred into 30% sucrose solution as described above, and sections were then mounted in anterior–posterior order in order to facilitate the determination of GRIN lens location for each mouse on an epifluorescent microscope.45, which utilizes a single reference frame and high-contrast features in the image to shift frames with motion to matching XY positions throughout the video. Black borders from XY translation in motion correction were cropped, and cell segmentation was performed using Constrained Non-negative Matrix Factorization for microEndoscopic data (CNMF-E)46. Motion corrected and cropped videos were ran through the CNMF-E algorithm with an estimated cell diameter of 18 pixels, and identified neurons were sorted by visible inspection for appropriate spatial configuration and Ca2+ dynamics consistent with signals from individual neurons. We report the non-denoised temporal traces extracted by CNMF-E as neurons’ temporal activity. These traces were z-scaled with an estimated Gaussian noise level, corresponding to a scaled version of ΔF/Fo of each neuron. Ca2+ transient events were then defined with an event detection algorithm in which transients were Z-scored . Ca2+ events were defined as transients exceeding a 2 s.d. amplitude from a 0.5 s.d. baseline, lasting a minimum duration /t_half], where Ao = 0.5 and A = amplitude of that transient; t_half for GCaMP6f was 200 ms, before returning to a 0.5 s.d. baseline level. Additional Ca2+ transient rising events within detected Ca2+ transients that were large and multi-peaked were then detected using the findpeaks function in MATLAB  with the following parameters . All detected Ca2+ transients were visibly inspected for each cell to verify accuracy.Calcium video image processing was performed using Mosaic and Inscopix Data Processing software . Videos were temporally downsampled to 5 frames per second, spatially downsampled by a binning factor of 4, and lateral brain movement was motion corrected using the registration engine TurboregXY translation. Motion correction accuracy was then determined by visually tracking cells across imaging days (using a hand-drawn ROI in ImageJ ROI Manager) to ensure that cell spatial locations did not shift throughout the entire concatenated video.In order to track the same cells across contextual conditioning days, videos from the three imaging sessions were concatenated into a single video prior to motion correction. The concatenated video was then motion corrected to a single reference frame to ensure that all frames were shifted to the same location during motion correction 2+ transient events and mouse behavior were analyzed with custom MATLAB  functions to calculate the rate of Ca2+ transients and correlated activity per cell while mice explored different contexts. Freezing behavior was scored using ObserverXT software, and behavior data was downsampled to five frames per second to match Ca2+ transient data sampling. Non-parametric statistical comparisons were applied to all analyses.Ca2+ transient amplitudes, temporally binned into one second time bins, and the Pearson’s correlation coefficient was computed for each pair of neurons. An R threshold of 0.3 was applied to this matrix to reduce bias from noise. Next, a graph was constructed using the NetworkX python package47 to visualize and quantify the higher order structure of the neural correlations31. The correlated pair ratio  and clustering coefficient (# edges among neighbors/# possible edges among neighbors) were computed for each neuron. Due to the sparse nature of these neural graphs, we also determined whether each neuron was a member of a connected component containing at least two other neurons. A connected component is defined as a subgraph of nodes connected to each other by paths but not connected to any other nodes. Each neuron was assigned either a 0 or 1 indicating whether it was a component member, and the proportion of neurons in a component (component probability) was computed for each imaging session.For all correlated activity analysis, only mice with ≥20 cells per FOV were included in the analysis given the sampling limitation of potential correlated pair partners in sparse FOVs  and calculated mock Pearson’s R values from the shuffled calcium events to create a shuffle distribution of R values for each cell pair. R thresholds at p < 0.05 from the shuffle distribution were then applied to determine the number of correlated pairs with shuffled R thresholds and compared to correlated pairs defined by R > 0.3 thresholds .We compared the 2+ activity heatmap during homecage optoLED pulses in vGAT-Chrimson mice was generated by taking the average calcium activity of Ca2+ transients per cell (non-transient time bins were pre-filtered to zero baseline) per 200 ms time bin from the 6 s preceding optoLED ON and the first 6 s of each 30 s optoLED ON pulse. The calcium activity across all cells was then normalized to the max and min calcium activity time bin, and cells were sorted by their rank in average change in Ca2+ activity from OFF to ON time bins.A Ca2+ events were shuffled in time for individual cells (1000 iterations), and shock or tone rates were re-calculated from those behavioral time periods to generate a null distribution of shock or tone Ca2+ event rates for each cell. A cell was considered a shock-cell or tone-cell if its Ca2+ event rate during those periods exceeded a 1 SD threshold from the null distribution.For defining cells that were significantly responsive to shocks or tones, Ca2+ events were shuffled in time for individual cells (1000 iterations), and optoON shuffled rates were re-calculated from the first 6 s of each 30 s pulse  to generate a null distribution of optoON Ca2+ event rates for each cell. A cell was considered an opto-stim cell if its Ca2+ event rate during the optoON periods exceeded a 1 SD threshold from the null distribution. The threshold was determined by comparing Ca2+ rates for neutral cells defined at different thresholds, and the threshold at which neutral cells showed no significant Ca2+ event rate difference between optoON and optoOFF time periods was selected.For defining cells that were optogenetically stimulated by the optoLED during a homecage 30 s pulse session, Cap-value for the A2 shock-partner population was then calculated relative to the change in these random sample distributions.In order to control for a potential selection bias in our A2 shock-partner analysis , we randomly sampled a matching proportion of cells from the A2 non-shock-partner population with at least one correlated pair on A2 , and created a histogram of the change in graph parameters from A2 to A1 from each randomly sampled cell population (Supplementary Fig. Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Alzheimer’s disease can be modelled by different transgenic mouse strains. To gain deeper insight into disease model mechanisms, the previously described Tg4–42 mouse was analysed for transgene integration. On RNA/DNA level the transgene integration resulted in more than 20 copy numbers and further caused a deletion of exon 2 of the retinoic acid receptor beta. These findings were also confirmed on protein level with highly decreased retinoic acid receptor beta protein levels in homozygous Tg4–42 mice and may have an impact on the previously described phenotype of homozygous Tg4–42 mice to be solely dependent on amyloid-ß 4–42 expression. Since hemizygous mice show no changes in RARB protein levels it can be concluded that the previously described phenotype of these mice should not be affected by the retinoic acid receptor beta gene knockout. In order to fully understand the results of transgenesis, it is extremely advisable to determine the genome integration site and the basic structure of the inserted transgenes. This can be carried out for instance by next-generation sequencing techniques. Our results thus suggest that a detailed characterization of new disease models using the latest genomics technologies prior to functional studies could be a valuable tool to avoid an unexpected genetic influence on the animals’ phenotype that is not only based on the inserted transgene. This would also significantly improve the selection of mouse models that are best suited for therapeutic development and basic research. To gain deeper insight into the underlying disease mechanisms the use of AD animal models in research is highly relevant and thus far without alternative3. While cognitive deficits, amyloid deposits or neurofibrillary tangles and inflammation are observable in many AD mouse models, neurodegenerative pathology as characterized by e.g. hippocampal neuron loss is only observed in a few models9. For this reason, we further characterized the previously described Tg4–42 mouse model, as it develops neuronal loss and therefore represents a promising model for the development of new drugs or drug efficacy studies10. Homozygous Tg4–42 mice are characterized by an age depended, massive CA1 pyramidal neuronal loss in the hippocampus. Furthermore, synaptophysin staining in the CA3 region of the hippocampus demonstrates a modified synapse pattern in these mice, which might be caused by a possible disturbance of the neuronal network. Homozygous Tg4–42 mice exhibit no significant target quadrant preferences in the Morris water maze behaviour test, which indicates severe impairments in spatial memory. According to AD pathologies these mice show age-dependent impaired glucose metabolism in all cortical areas which were analysed by 18F-FDG-PET11. In contrast hemizygous Tg4–42 mice still demonstrate a significant preference for the target quadrant like wild type mice in the Morris water maze behavioural test at the same tested age, indicating no or only weak impairments in spatial memory. Moreover, also synaptic chances were less pronounced in hemizygous compared to homozygous mice12. These analyses reveal that the gene-dosage influences the phenotype of these mice. Homozygous mice develop enhanced neuropathological features and neuronal loss compared to hemizygous animals at the same age13.Alzheimer’s disease (AD) is the most common neurodegenerative disorder and mainly characterized by progressive neurodegeneration as well as extracellular accumulation of amyloid-ß protein (Aß) that is surrounded by dystrophic neurites and neurofibrillary tangles7. A weekly treatment with the full-length or Fab fragment of a monoclonal antibody directed against Aß4–42 also showed the ability to completely rescue spatial reference memory deficits and significantly stabilized neuron numbers in 6 month old homozygous Tg4–42 mice10.Two different studies exhibited a rescue of this phenotype by therapeutic interventions. Homo- and hemizygous Tg4–42 mice that were allowed to exercise in enriched housing conditions revealed improved performance on the balance beam, rotarod and rescued performance in the Morris water maze test. Additionally, this increased physical activity caused decreased neuron loss which resulted in a higher number of CA1 pyramidal cellsTo further characterize the genetic features of Tg4–42 mice, whole genome sequencing was performed to determine the integration site(s) of the transgene and estimate the transgene copy number as well as assess the presence of structural variants surrounding the transgene integration site and the transgene sequence itself. Decreased expression and reduced protein levels of the retinoic acid receptor beta in brain tissue of homozygous Tg4–42 mice was found, questioning the cause of the previously described phenotype in homozygous Tg4–42 mice to be solely dependent on Aß4–42 expression.Targeted Locus Amplification of the Tg4–42 + /+ mouse model showed that the Aβ4–42 transgene is inserted in more than 20 copies of head to tail orientation at position 17,431,476–17,651,381 (mouse genome assembly mm9) on chromosome 14 (see Supplementary Information). Only the last transgene copy is in reverse orientation. Furthermore, exon 2 of the retinoic acid receptor beta (RARB) gene was deleted in Tg4–42 mice Fig. .Figure 1To analyse how this genetic deletion influences RARB RNA expression levels and if expression of whole mRNA is affected, primer pair 1 which binds between exon 5 and 6 was designed. Quantitative PCR using primer pair 1 showed expression levels that were almost reduced to 50% in hemizygous Tg4–42 + /− and almost immeasurable in homozygous Tg4–42 + /+ animals Fig. . To valiTo confirm these results, two further primer pairs were used binding from exon 2 to 3 and from exon 7 to 8 and thus outside of the deleted gene region of RARB Fig. .qPCR was further performed using cDNA of three different homozygous Tg4–42 + /+ and three wild type (WT) striatal samples. Results show that all three primer pairs showed a significant knockdown of RARB RNA levels in all Tg4–42 + /+ animals Fig. .To confirm the gene expression results on protein level Western blot analysis was performed. Many different approaches like two different protein extraction methods, different protein concentrations, three different primary antibodies , different incubation settings and different gel detection methods were used but did not show satisfactory results because of the non-specificity or low sensitivity of used antibodies (data not shown).Furthermore, protein analysis with Mass spectrometry was carried out in an untargeted as well as targeted approach but the concentration of RARB protein in WT animals was under the limit of detection.As the traditional Western blot as well as the mass spectrometry analysis did not show any useful result on protein level, Simple Western blot analysis was performed. Therefore, a monoclonal retinoic acid receptor beta 2 antibody  already unsuccessfully used for classical Western blotting was tested in different concentrations. The antibody showed a specific signal at 48 kDa in WT animals which decreased at higher antibody dilutions. Moreover, the specificity control (SP-CTRL) without a biological sample, to evaluate if the primary antibody sticks to capillary wall material and a negative control (N-CTRL) without the primary antibody to check for cross-reactivity, showed no unspecific signal Fig. . For fur14.In this study we used the Tg4–42 mouse model which was previously introduced and described to express N-truncated amyloid-β 4–42. Homo- and hemizygous Tg4–42 animals develop a typical AD phenotype, including neuronal loss and spatial learning deficits albeit without plaque formation16. For further validation of the genomic integration site, Sanger sequencing could be performed, although Baudhuin and colleagues question the necessity of this confirmation17. To clarify the impact of transgene insertion on the protein level, we used the Simple Western blot system. This novel capillary electrophoresis-based Western blot technology requires much smaller amounts of tissue than traditional Western blot which is important if limited sample amounts are available. Furthermore, the higher sensitivity as well as reproducibility and excellent quantification options by analysis of the Area Under the Curve are big advantages which led to the decision to use the capillary Western approach in our study19. The Simple Western blot system analyses resulted in a highly decreased RARB protein level in homozygous Tg4–42 mice. Thus, the integration of the Aβ4–42 transgene appears to substantially disrupt RARB gene regulation, resulting in a knockdown of this gene and in turn downregulation of RARB protein. The described phenotype of the homozygous Tg4–42 mouse model could therefore be caused by the Aβ4–42 transgene or by RARB knockout20.Using qPCR, we have shown the transcriptional impact of transgene integration in homozygous Tg4–42 mice. Whole genome sequencing located the integration site of the transgene on chromosome 14, which caused a deletion of exon 2 of the retinoic acid receptor beta. TLA combined with next generation sequencing is best suited for a robust detection of integration sites or transgene sequence variants22. The retinoic acid receptor beta deficiency eliminates hippocampal CA1 long-term potentiation and causes severely impaired spatial learning in the Morris water maze behavioural test, like it is described in homozygous Tg4–42 animals. In contrast, hemizygous Tg4–42 animals still show target zone preferences, which might also be attributed to unaltered RARB protein levels. These generated RARB knockout mice are viable, fertile and display no obvious defects23. Nevertheless, it is described that a loss of both wild type alleles of the RARB gene leads to a cell deficit on the ganglion cell population, which of course can influence the phenotype24. Otherwise, therapeutic intervention studies with a monoclonal antibody or with enriched housing conditions were able to reduce hippocampal neuron loss and showed a rescue of spatial reference memory deficits in homozygous Tg4–42 mice. These data would rather indicate that the observed phenotype of homozygous Tg4–42 mice is caused by the Aβ4–42 transgene10. Another conclusion from the current work is that it should be obligatory to analyse a possible treatment effect in more than one Alzheimer’s disease mouse model. Since hemizygous mice showed no changes in RARB protein levels, the previously described phenotype of these mice should not be affected by the retinoic acid receptor beta gene knockout. Our results demonstrate that it is extremely advisable and helpful to determine the genomic integration site and the basic structure of the inserted transgenes for instance by next-generation sequencing techniques to be able to fully understand the effects of transgenesis. In order to avoid an unexpected genetic impact on the animals’ phenotype, which is not only based on the inserted transgene a detailed characterization of new disease models using the latest genomics technologies is of enormous importance. This would also significantly improve the selection of mouse models that are best suited for basic research and therapeutic development.A generated RARB null mutant mouse model described in the literature with a disruption of all isoforms of RARB shows a very similar phenotype compared to homozygous Tg4–42 miceIn conclusion, our data provide detailed information about the integration site of the Aβ4–42 transgene and additionally show decreased expression and reduced protein levels of the retinoic acid receptor beta in brain tissue of homozygous Tg4–42 mice. These results question the cause of the previously described phenotype of homozygous Tg4–42 mice to be solely dependent on Aß4–42 expression. Further studies need to address this issue in detail.ad libitum in an AAALAC accredited animal facility. All animal studies adhered to the Austrian law for the care and use of laboratory animals.Male homozygous (Tg4–42 + /+), hemizygous (Tg4–42 + /−) and non-transgenic wild type (WT) control mice were bred and housed in individually ventilated cages in a controlled environment on a 12/12-h light/dark cycle (lights on from 6 a.m.–6 p.m.), with humidity between 40 to 70%, temperature of approximately 21 °C and food and water Mice were deeply anesthetized by i.p. injection of 600 mg/kg pentobarbital at the age of 6 months and transcardially perfused with physiological (0.9%) saline. Thereafter, brains were removed, hemisected and subdivided into different brain areas, immediately frozen on dry ice and stored at −80 °C.et al.15. PCR products were purified and library prepped using the Illumina NexteraXT protocol and sequenced on an Illumina Miseq sequencer. Reads were mapped using BWA-SW, which is a Smith-Waterman alignment tool. This allows partial mapping which is optimally suited for identifying break spanning reads. For mapping the mouse genome, version mm9 (MGSCv37) was used by this company.Targeted Locus Amplification (TLA) of Tg4–42 mice was performed by Cergentis  as previously descried in de Vree Total striatal RNA was extracted using peqGOLD TriFast Kit . 1 µg of total RNA was reverse-transcribed into single stranded cDNA using iScript gDNA Clear cDNA Synthesis Kit . Quantitative PCR (qPCR) was performed with Bio-Rad CFX Connect thermo cycler using Takyon No Rox SYBR MasterMix dTTP Blue  with the primers  listed in Table All PCR amplifications were performed in a total volume of 20 µl, containing 2 µl of cDNA, 10 µl of 2x Takyon SYBR mix, 1 µM of each primer and 6 µl of aqua bidest. The amplification protocol consisted of an initial denaturation at 95 °C for 3 min, followed by 39 cycles of denaturation at 95 °C for 0.2 min, annealing at 60 °C for 0.5 min and extension at 72 °C for 0.5 min.Mouse striatal samples were weighted and lysed in 15 volumes RIPA buffer containing protease- and phosphatase-inhibitor and sonicated with the Ultra-Turrax for 30 s. Afterwards tissue homogenates were centrifuged at 20,000 × g at 4 °C for 10 min to remove insoluble material. The protein concentration of tissue homogenates were determined using Pierce BCA protein assay kit . For capillary simple Western blotting homogenates were diluted to 0.65 mg protein/ml sample, using sample buffer.Samples were prepared and analysed according to the manufacturer’s instructions . Briefly, four volumes of sample were mixed with one volume of fluorescent 5xMaster Mix (Protein Simple) and denatured at 95 °C for 5 min. Primary antibodies against retinoic acid receptor beta 2  and Glycerinaldehyd-3-phosphat-Dehydrogenase  were diluted 1:10 (RARB) and 1:100 (GAPDH) in antibody diluent 2. The samples, biotinylated ladder, primary antibodies, secondary antibodies, chemiluminescent substrate, Stacking Matrix 2 and the Separation Matrix 2  were dispensed into the assay plate. After adding Simple Western assay buffers subsequent separation, immunodetection and analysis steps were performed automatically and digitally by the Simple Western blot System.Compass for SW software (Protein Simple version 4.0.0) was used to visualize Simple Western lanes digitally, automatically analyse signal peaks and calculate the area under the curve of the peak of interest.All statistical analyses and graphs were performed using GraphPad Prism 7.0 software .All experiments including animal tissue were performed in accordance with the Austrian guidelines for the care and use of laboratory animals . Animal housing and euthanasia were approved by the Styrian government .Supplementary information.


Our results show that water oxygen isotopic composition (δ18Owater) rather than lake summer water temperature (Twater), is the main determinant of δ18Ocarb, irrespective of whether oxygen isotope equilibrium is achieved. There are significant linear correlations between δ18Ocarb and elevation, as well as that between δ18Ocarb and latitude for lakes located on the Tibetan Plateau. In Western China, the spatial distribution of δ18Ocarb is consistent with that of δ18Owater, and is ultimately controlled by the isotopic composition of local precipitation (δ18Oprecipitation) that depends on the source of water vapor. Therefore, changes in δ18Ocarb can be predominantly interpreted as variations of δ18Owater, which in turn represent changes in δ18Oprecipitation for paleoclimate reconstructions in this region, and may be relevant to studies of other areas.In the carbonate-water system, at equilibrium, the oxygen isotopic composition of carbonate is dependent not only on the temperature but also on the isotopic composition of host water in which the carbonate is formed. In this study, lake surface sediment and water samples were collected from 33 terminal lakes in Western China to evaluate controls on the oxygen isotopic composition of lacustrine authigenic carbonates (δ In systems that are in thermodynamic equilibrium, an increase in temperature with increased atomic vibration frequency leads to decrease in isotopic fractionation between the reactant water and the carbonate mineral that precipitates from it5. Accordingly, a number of empirical relationships between carbonate oxygen isotopic composition and temperature have been established8, and are widely applied to lakes in paleoclimate studies to reconstruct water temperature11.The carbonate oxygen isotope geothermometer has been one of the most important tools for reconstructing paleotemperatures since it was established by Urey12. The assumption of equilibrium has not been tested in detail in modern lacustrine authigenic carbonates and there are discrepancies relating to equilibrium fractionation factors and kinetic fractionation processes among theoretical calculations, experimental measurements, and modern natural observations14. In slowly precipitating experiments from Kim and O'Neil7, the authors judged the smallest fractionation factors to be the best representations of equilibrium fractionation factors, despite the fact that fractionation factors increased with increasing initial concentrations of metal ion and bicarbonate at a certain temperature. However, Zeebe15 argued that the observed effects by Kim and O'Neil7 were actually equilibrium fractionations expressed at different solution pH values. Based on Devils Hole calcite deposition, which is considered to be the most likely to approach to true thermodynamic equilibrium, Coplen16 considered that formerly adopted oxygen isotopic fractionation factors might be underestimated when used in natural samples because of the restriction of experimental conditions and theoretical calculation methods. Analysis of clumped isotopes in a series of samples led the authors to conclude that most carbonates precipitate out of equilibrium17. A study by Watkins et al.18 first isolated the kinetic oxygen isotope effects occurring at the calcite-water interface and analyzed the effects of changing calcite precipitation rates and water pH. Recent studies from Devriendt et al.19 and Guo et al.20 also confirmed that different solution pH values, crystal growth rates, and mass-dependent processes might lead to disequilibrium fractionations. In addition, most laboratory experiments and theoretical calculations are based on limiting multiple external conditions with only one variable; however, natural settings are more complex. This may result in inconsistencies of calculated oxygen isotope fractionation factors among different laboratories, as well as that between natural records and laboratory experiments21. Co-variation between lake carbonate oxygen and carbon isotopes have been used to identify lake systems where kinetics dominate22. Therefore, an accurate determination of whether equilibrium fractionation occurs, and an understanding of the processes that control kinetic fractionation are essential for the accurate interpretation of carbonate oxygen isotopic composition as an environmental proxy.Our work on oxygen isotope systematics in modern lake carbonates is motivated by three issues. First, the fundamental precondition to applying an isotopic geothermometer is that carbonate precipitating from water is in oxygen isotope equilibrium5. Prior work relies on an assumption of the lake water oxygen isotopes or numerical modeling23. In addition, some field studies show that the oxygen isotopic compositions of lacustrine carbonates are largely influenced by rainfall or local precipitation/evaporation balance, rather than temperature changes25.Second, the oxygen isotopic composition of lake water for geologic samples is often difficult to constrain. In many paleoclimate reconstructions, where it is often assumed carbonate precipitation is in near equilibrium, there are often no independent constraints on either temperature or water isotopes27. However, there are few studies that have examined the modern oxygen isotopic systematics of authigenic carbonates in lakes; most have focused on investigations of paleoenvironments30. Therefore, an understanding of whether lacustrine authigenic carbonates can achieve oxygen isotope equilibrium when they were precipitated in modern natural lake settings, and understanding of which factors, such as water temperature, precipitation, lake elevation, latitude or longitude, could be reflected by the oxygen isotopic composition of lacustrine authigenic carbonates collected from a certain region is essential for accurate interpretation for paleoclimate reconstructions.Third, lakes play a pivotal role as recorders of climate change, and lacustrine authigenic carbonates are an ideal material for isotope analyses utilized in a number of studies31. There are a large number of natural lakes distributed in Western China, which account for nearly 58.3% of the number and 66.9% of the area of all Chinese lakes32. Due to the influence and limitations of complex geological formations and natural environments, most of the lakes are terminal lakes with relatively high salinities35. Based on the large differences of geographical locations, basin topography, local climates, and water hydrology conditions, these lakes are sensitive to environmental changes, especially to variations in temperature and humidity36.Western China is considered to be a semi-arid and arid region, characterized by low, uneven rainfall and high evaporationIn this study, we present measurements from modern lacustrine authigenic carbonates and lake surface water samples collected from thirty-three terminal lakes in Western China ranging in elevation and temperature Fig. . We alsoLMSW) from June to August was calculated using the data recorded by the on-site data loggers retrieved from twelve lakes (Supplementary Table MTW), measured manually using a mercurial thermometer in the field, and the TLMSW from the loggers that were able to be retrieved from the lakes:Logged Mean Summer Water Temperature  by applying Eq.  is either TLMSW (lakes with loggers) or TCMSW (lakes without loggers). Twater ranged from 9.8 to 25.6 °C  ranged from − 8.82 to 5.65‰ (VSMOW) and from − 79.12 to 11.22‰ (VSMOW), respectively  value of calcite, aragonite, or dolomite exceeds 0 for each sample (Supplementary Table 37. X-ray powder diffraction (XRD) analyses show that there are nine pure calcite samples and twenty-four mixed mineralogy samples in this study (Supplementary Table 18Ocarb) was calculated based on a stable isotope mixing model. δ18Ocarb spanned a relatively large range with a minimum of − 9.61 ‰ (VPDB) and a maximum of 3.77‰ (VPDB) between lakes. The oxygen isotope fractionation factor between carbonate and water (1000lnα(carb−water)) ranged from 22.38 to 32.71. Carbon isotopic composition of lacustrine authigenic carbonate (δ13Ccarb) ranged from − 4.06 to 4.97‰ (VPDB)  and Twater:As shown in Fig. (carb−water) values cannot be used to infer lake summer water temperature.All points are scattered on both sides of the fitted line, where 18 points distribute out of the 95% confidence interval. Thus, an initial inspection indicates the 1000lnα18Ocarb with δ18Owater shows a significant correlation:The comparison of δ18Ocarb exhibit a strong positive correlation with δ18Owater, with higher δ18Owater corresponding with more enriched δ18Ocarb  and δ18Ocarb ranged from − 19.9 to 5.65‰ (VPDB). There is a statistically significant linear correlation between δ18Ocarb and δ18Owater from combined isotope data in different sections  on lake elevation (Elev) on the Tibetan Plateau. But the Elev/δ18Ocarb coefficient is not very significant  for the Northwestern Xinjiang and Inner Mongolia regions where lake elevation below 3000 m. On the Tibetan Plateau, there is a positive linear correlation between lake latitude and δ18Ocarb   in Western China  Fig. c. But itina Fig. e. In genues Fig. .Figure 55. In this section, we discuss the effects of these two factors respectively and spatial distributions of the oxygen isotopic composition of lacustrine carbonates in Western China.Two critical factors controlling the oxygen isotopic composition of carbonate mineral are the temperature of carbonate formation and the oxygen isotopic composition of the solution from which carbonate minerals precipitate41. In general, lacustrine authigenic carbonates precipitate in summer, when the carbonate saturation of lake water peaks and carbonate solubility is simultaneously depressed in the epilimnion43. Variations in water temperatures averaged over several summers in recent years usually less than ± 1 ~ 2 °C at a certain lake, especially for the lakes located on the Tibetan Plateau45. Therefore, the measured summer water temperatures could represent the temperatures when authigenic carbonate samples were precipitated. In large closed lake systems, variations in water isotope composition caused by precipitation or evaporation are usually homogenized by buffering of large lake volume24. Therefore, a large lake with long water residence time could ‘average out’ short-term changes in isotope compositions and instead reflects relatively long-term isotope compositions under similar climate and hydrological conditions47. As the size of sampled thirty-three lakes is relatively large, and the relative humidity in Western China has not changed greatly in recent years49, variations in water isotope values during the course of one or several summers may not significant50. Because we do not have longitudinal data on lake water isotope values at our lakes, we assume that the measured isotope values of water samples that collected at the lake center and at the same time with sediment samples could be considered as long-term average compositions during the summer when authigenic carbonates were precipitated.In this study, the carbonate samples provide integrated climate signals cover several years as the sedimentation rate of surface sediment ranges from 0.01 to 0.3 cm/yr throughout our sampling locations2. However, there is no statistically significant correlation between the 1000lnα(carb−water) and water temperatures in natural lake settings in this study  by applying published temperature calibrations to the 1000lnα(carb−water) values of our lakes, and compared modeled TCW with independently measured Twater. As shown in Fig. CW ranges from 3.8 to 68.2 °C, which is far beyond Twater (9.8–25.6 °C) of our lakes. The slope and intercept of 1000lnα(carb−water)-TCW regression lines are lower than that of 1000lnα(carb−water)-Twater regression line . This suggests that factors other than temperature are contributing to oxygen isotope fractionation observed in lacustrine authigenic carbonates in this study. The discrepancies between our results and previous studies may be attributed to carbonate disequilibrium precipitation.For equilibrium carbonate precipitation, oxygen isotope fractionation is directly controlled by thermodynamics, and the isotope fractionation factor is a function of temperatureudy Fig. a. Since 51. As lake water is the largest reservoir of oxygen isotopes for DIC species and precipitated carbonates, we employed a mass balance calculation52 to calculate the expected oxygen isotope fractionation factor between DIC and water 2(aq) because there should not be a substantial influence of CO2 degassing on the DIC pool in natural lake settings53. The mass balance equation is:X denotes the molar fractionation of the DIC species which is determined by PHREEQC v. 2.18.00. software program54, and 1000lnαi denotes the individual fractionation factor between the DIC species and lake water reported by Beck et al.51 at a certain summer water temperature in each lake.Lacustrine authigenic carbonates form in a mixture of dissolved inorganic carbon (DIC) species in lake water. At equilibrium, the oxygen isotopic composition of precipitated carbonate closely approximates that of DIC at a certain temperature(carb−water) values to expected equilibrium (carb−water) values and the expected equilibrium P = 0.2033). Most of the 1000lnα(carb−water) values are lower than the expected equilibrium 18O values in natural lake settings.We compared our experimental 1000lnα55. With respect to lakes, there are two main factors that can lead to the disequilibrium fractionation that we observe; these may be complicated by multiple environmental controls and processes at a given site.Carbonate disequilibrium precipitation may be influenced by multiple mixed kinetic fractionation processes that originate from kinetic fractionations during the exchange of oxygen isotopes between water and DIC species, or between DIC species and carbonate, or a combination of both of these factors57. The oxygen isotope fractionation between water and DIC, as well as that between DIC and precipitated carbonates, will decrease with increasing pH, as the dominant DIC species changes from CO2(aq) to 55. Given that pH values differ between the lakes in this study, we evaluated the pH effect using a model from Watkins et al.55  as in this study. Since the growth rates of carbonate samples in this study are, as with many carbonates, likely to be higher than that of slowly precipitated calcites collected from the Devils Hole and Laghetto Basso, the time for oxygen exchange between the DIC and water might be insufficient to attain the equilibrium fractionation17. In this case, the DIC species with 16O isotopologues tend to preferentially participate in oxygen isotopic exchange, leading to the oxygen isotope composition of formed carbonates being lighter than theoretical values55. Therefore, kinetic fractionations caused by pH effect and growth rate effect might lead to large deviations of fractionation factors between expected equilibrium and measured results in natural lake settings, resulting in the lack of a significant correlation between 1000lnα(carb−water) and Twater in this study.Second, the oxygen isotope exchange between DIC species and water is a rate-limiting step for equilibrium18Ocarb and δ18Owater, we compiled more than five hundred oxygen isotope values of different types of carbonates from published papers  and in lake surface sediments . These results indicate that higher δ18Owater values correspond to higher δ18Ocarb values, even though hydrologic conditions differ between records and may be complex, and oxygen isotope equilibrium is not necessarily attained during carbonate precipitation. The positive correlation between δ18Owater and δ18Ocarb can be explained by the exchange of oxygen isotopes during the formation of carbonate4:18O is transferred from reactant H2O to precipitated CaCO3 minerals.In order to compare the relationship between δers Fig.  online. 18Owater, water pH and Twater were used in boosted regression tree (BRT) analyses to produce models that could estimate contributions of lake water parameters to δ18Ocarb for our sample sites. The BRT results estimate that δ18Owater, pH, and Twater account for 80.1%, 12%, and 7.9% of the variance in δ18Ocarb, respectively  between δ18Ocarb and δ13Ccarb for the lakes in this study. But if we classify samples by the location of lakes, the correlations between δ18Ocarb and δ13Ccarb become more considerable. Linear correlation coefficients (r) range between 0.35 and 0.92 depending on the location of the lake  and local precipitation (δ18Oprecipitation and δDprecipitation) in Western China, and investigated influences of lake latitude, elevation, and lake water salinity on δ18Owater and δDwater in detail. In their results, isotope enrichment by local evaporation, of differing magnitudes depending on location, is also recorded by the Local Evaporation Line (LEL) that shifts to the right of the Local Meteoritic Water Line (LMWL)  Fig. c. Howeve18Owater dominate the variations in δ18Ocarb, the spatial distribution of δ18Ocarb could be inherited from that of δ18Owater in Western China. Based on Rayleigh fractionation, isotope values for precipitation become gradually depleted when water vapor climbs high mountains66. As shown in Fig. P < 0.0001) and 0.88 (P < 0.0001) for carbonate and lake water, respectively, demonstrating that altitude effect is significant on the Tibetan Plateau. But δ18Ocarb variances caused by the altitude effect is not significant  at the Northwestern Xinjiang and Inner Mongolia regions where lake elevation is below 3000 m. On a worldwide scale, water vapor originates from the tropical ocean65. The latitude effect usually results in heavy oxygen isotope depletion of precipitation when water vapor is transported from southern to northern regions67. Although δ18Owater and δ18Ocarb are negatively correlated to elevation, they are positively correlated to latitude for lakes located on the Tibetan Plateau. It indicates that the variation of δ18Owater and δ18Ocarb may not be explained by the latitude effect in Tibetan Plateau. For the southern Tibetan Plateau, precipitation with negative isotope value is mainly originated from the Bay of Bengal and the Arabian Sea68. But the oceanic water vapor is usually blocked by the Himalayas and Tanggula Mountains and can hardly arrive northern part of the plateau69. Lakes located on the northern Tibetan Plateau are under the control of dry continental air masse with enriched heavy isotopes64. As a result, δ18Owater and δ18Ocarb values of lakes located on the northern Tibetan Plateau are higher than these of lakes located on the southern Tibetan Plateau. Since δ18Oprecipitation values provided by the westerlies and East Asia summer monsoon are different67, the insignificant Lat/δ18Ocarb coefficient  for lakes located in the Northwestern Xinjiang and Inner Mongolia regions could be also attributed to different vapor sources. As shown in Fig. 18Ocarb  and δ18Owater  are insignificant in Western China where precipitations are originated from different water sources. It implies that there is not a continental effect that causes a general shift towards lower δ18O values in lake water and precipitated carbonates from east to west in Western China. Therefore, different water vapor sources contribute to the distinct spatial distribution of δ18Ocarb for lakes located in different regions in Western China.As changes in δ18Ocarb can be predominantly interpreted primarily as δ18Owater, which in turn indirectly reflects changes in δ18Oprecipitation. The spatial variations of δ18Ocarb in Western China are ultimately controlled by water vapor source.In conclusion, although disequilibrium fractionation occurs during authigenic carbonate precipitation, the δ71. It is likely that the crystallization and precipitation of lacustrine carbonate occurs under non-equilibrium conditions17. As such, even though temperature is an essential factor controlling the oxygen isotopic composition of carbonate minerals, it is questionable to reconstruct water temperature using lacustrine authigenic carbonates, without a detailed discussion of whether isotopic equilibrium was attained. In contrast, our results indicate that the isotopic composition of lacustrine authigenic carbonate depends on that of host water, regardless of whether the isotope equilibrium conditions are reached. Therefore, in paleoclimate reconstructions, changes in oxygen isotopic composition of lacustrine authigenic carbonate from terminal lakes in Western China can potentially be interpreted as the oxygen isotopic composition of lake water, which in turn indirectly reflects variations of the oxygen isotopic composition of precipitation, assuming intra-annual changes in temperature were relatively small. Furthermore, the spatial distributions of lacustrine authigenic carbonate oxygen isotope values could reflect different water vapor origins in Western China.In natural lacustrine settings, the temperature dependent oxygen isotope equilibrium fractionation between lacustrine carbonate and lake water can be complicated by multiple environmental controls and processes, such as ionic saturation, DIC speciation, growth rate, and other factors18Ocarb and 1000lnα(carb−water) in natural lake settings in Western China; (2) Many factors may lead to kinetic oxygen isotope fractionation during authigenic carbonate precipitation in lacustrine settings, including pH and growth rate-related effects. These factors can account for a larger fraction of the variance in δ18Ocarb and 1000lnα(carb−water) in these samples; (3) A positive correlation between δ18Owater and δ18Ocarb is observed in the 33 lakes located in Western China. δ18Owater is the dominant factor governing δ18Ocarb, regardless of whether the isotope equilibrium conditions are reached during the precipitation of authigenic carbonates; (4) The spatial distribution of δ18Ocarb is consistent with that of δ18Owater and δ18Oprecipitation, and is ultimately controlled by water vapor source in Western China; (5) Under either the presence or absence of isotope equilibrium, changes in δ18Ocarb from terminal lakes in Western China can be predominantly interpreted as variations of δ18Oprecipitaion, instead of changes in temperature. This provides an important basis for future paleoclimatic reconstructions using the carbonate oxygen isotope proxy in lacustrine authigenic carbonates.Overall, the main conclusions we reached are: (1) Temperature is associated with a relatively small fraction of the observed variance in δIn July and August 2016, surface sediment and water samples were collected from thirty-three lakes located in Western China. To ensure that the samples were not influenced by hydrological or human disturbance, samples were collected at the lake center for smaller lakes and at least 2 km away from the shore for larger lakes.In each lake, the upper most 0.5 cm of surface sediments were collected using a stainless grab and were placed in leak proof plastic bags. At the same site, surface water samples were collected at a depth of ~ 50 cm below the water surface. Water samples were collected and stored in 500 ml high-density polyethylene (HDPE) bottles which were initially washed three times using the lake water. The bottles were completely filled with water samples and sealed with a cap secured with plastic electrical tape to avoid evaporation or any isotopic exchange with air. Sediment and water samples were kept cool in the field and were then stored at 4 °C in Capital Normal University, China.MTW was measured once for each lake at around 2 p.m., during the warmest time of day, when the sediment and water samples were collected in the field. TMTW was manually measured using a mercurial thermometer at 50 cm below the water surface in the same location of water sampling. HOBO U22 Water Temperature Pro v2 data loggers were also set at a depth of 50 cm below the water surface of each lake. Temperature data was collected at 15-min intervals over the course of one year. We returned in the following summer and successfully retrieved 12 data loggers, while the rest of the loggers were lost.T+, Ca2+, Mg2+, −, 72. Cl− concentrations were determined by AgNO3 potentiometric titration, with a precision of ± 0.1%. 4. Concentrations of K+ were measured by gravimetric methods through precipitation of potassium tetraphenylborate [KB(C6H5)4]. Ca2+ and Mg2+ concentrations were measured by ethylene diamine tetraacetic acid (EDTA) titration with errors of ± 0.5%. Na+ concentrations were calculated by charge balance:54 with the speciation model wateq. database.Lake surface water samples were analyzed for K18Owater and δDwater were conducted at the Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, China using an LGR DLT-100 Liquid Water Isotope Analyzer . Calibration of the measurements used three internal LGR standards . δ18Owater and δDwater were reported relative to VSMOW. The measurement accuracy was typically better than ± 0.1‰ for δ18Owater and ± 0.5‰ for δDwater.δ73. Only fine sieved fractions smaller than 45 μm were collected, frozen in a refrigerator overnight and then vacuum freeze-dried for 48 h using the Boyikang FD-1A-50 Freeze Dryer at approximately − 50 °C (30 Pa), until the samples were dried. Around 2 g of each sieved sediment was ground using agate mortar and pestle, and stored in a desiccator.The wet surface sediment samples were soaked in deionized water for about 2 h and then wet sieved with a 350-mesh (45 μm) sieve. Materials exceeding 45-μm containing detrital mixtures and biogenic carbonates (containing primarily ostracods) were filtered outα radiation (λ = 1.5406 Å). The diffraction spectral pattern was measured at a scanning rate of 2° min-1 for 2θ ranging from 10° to 80°. Mineral identification and semi-quantitative analyses were estimated from the bulk mineral diffractograms using the reference-intensity ratio (RIR) matrix-flushing method76 aided by the use of an automated search-match computer program X’pert HighScore Plus. The uncertainty of this semi-quantitative analysis was approximately ± 5% (1σ).Around 0.5 g of sediment powder were loaded into a plastic sample holder and the surface of the powder was smoothed prior to XRD measurements that were performed at the Qinghai Institute of Salt Lakes, Chinese Academy of Science, China, using a Phillips X-pert Pro X-ray diffraction with Cu K2O2 for 4 h to remove any remaining organic material. Resulting samples were collected on a 0.45 μm cellulose nitrate filter membrane and oven-dried at 40 °C. Depending on instrument sensitivity and carbonate content, the amount of sample used for isotope analyses varied between 12 and 95 mg.The fine sieved sediment samples were treated with 3% H18Ocarb and δ13Ccarb were measured with a Thermo Scientific MAT 253 gas source isotope mass spectrometer at the University of California, Los Angeles, USA from 2017 to 2018. Samples were reacted with 105% phosphoric acid (ρ = 1.92 g/mL) for 20 min on a 90 °C online common phosphoric acid bath system to convert to CO2 gas for analyses. The liberated CO2 was successively passed through a dry ice/ethanol trap (− 76 °C) and a liquid nitrogen trap (− 196 °C) to remove water and other compounds. After the initial purification step, the CO2 was passed through silver wool to remove sulfur compounds and then passed through a Porapak Q gas chromatograph column at − 20 °C to remove any additional contaminants before being transferred into bellows of the mass spectrometer for analysis. Data were collected over 9 acquisition cycles to determine δ13C and δ18O. A high purity pre-calibrated CO2 tank was used as a reference gas , whose composition has been determined by Oztech through comparison with NBS standard gases and CO2 evolved by acid digestion from NBS-19 and NBS-18. At least three replicates per sample were performed.The δ18O/16O fractionation by phosphoric acid digestion at 90 °C was corrected using an acid fractionation factor of 1.0079577. For aragonite, an acid fractionation factor value of 1.00854 was calculated by extrapolating the relationship reported by Kim et al.78. For dolomite, we used an acid fractionation factor of 1.009379. For monohydrocalcite, we used the same fractionation factor as calcite80. For samples that are a mixture of two or three carbonate minerals as determined by XRD, weighted acid fractionation factors were calculated.For calcite, 18Ocarb and δ13Ccarb are reported on the VPDB scale. We ran NBS-19 standards. The average measured value for NBS-19 is: δ13C =  − 2.143 ± 0.021‰ VPDB, δ18O = 1.938 ± 0.012‰ VPDB (n = 6). We also ran ETH-1 to 4 as standards81. Carbonate standards were analyzed between every 2–3 samples and were prepared and analyzed in the same manner as samples.δa, b and c denote the content of calcite, aragonite and dolomite, respectively. We calculated δ18Ocarb using weighted fractionation factors to reduce uncertainties caused by mineral specific fractionation processes:18Ocarb' denotes raw carbonate δ18O results. x and y denote the content of aragonite and dolomite, respectively. faragonite and fdolomite denote the offset of δ18O between dolomite, aragonite and calcite at a certain water temperature in each lake82.Based on the stable isotope mixing model:18Ocarb data including laboratory synthetic carbonates, core top lacustrine sediments and other field samples, and δ18Owater from published papers. Synthetic carbonates were included within this study if their precipitation conditions were similar to natural settings .We synthesized δgbm.step function in the dismo package 1.1-4 version83 in R 3.6.284. We used a default bag fraction of 0.5, a Gaussian error distribution, a very slow learning rate (0.0005) and a tree complexity of 3. The local polynomial regression (LOESS) curves were fitted using the loess function in R with span at 0.75, to validate the obtained BRT curves.BRT analyses were employed to estimate contributions of the lake water parameters to lacustrine authigenic carbonate isotopic composition. All BRT models were generated using the Supplementary Information.


Post-treatment filters have also demonstrated successful recovery of vital nutrients by precipitating ammonium magnesium phosphate hydrate, a documented valuable slow-release solid fertilizer. These filters have a great potential for advancing access to improved sanitation while simultaneously increasing capacity for small-scale economic agricultural development in geographic regions lacking improved sanitation.Simple trickling nitrification filters and submerged denitrification filters were developed to provide post-treatment to high-strength human waste anaerobic digestate with the aims to (i) effectively recover nutrients in a useful form as a fertilizer and to (ii) treat digestate such that it could be reused as flush water in water scarce regions. The tested filter media  are low cost and sustainable materials and can be locally sourced where on-site sanitation facilities are in high demand. Experimental data from laboratory operation with digestate from anaerobic digestion of dog feces and human urine revealed that the filters achieved a combined removal of chemical oxygen demand (COD), total nitrogen (TN), and phosphorus (PO  •Low-cost filters were investigated for the treatment of fecal waste digestate.•84% of the COD was removed in the GAC medium trickling filter.•69% of total N was removed in the biochar medium trickling filter.•4-P was removed across the system.89% PO•Ammonium magnesium phosphate hydrate was precipitated in the denitrification filter. It is eHowever, the implementation of sewer systems in access-deprived areas presents many financial and logistical challenges. Also, the large amounts of water needed for such centralized systems makes them unfit for many places where water is scares. Many areas around the world are increasingly adopting decentralized on-site sanitation (OSS) technologies. Currently, OSS facilities provide sanitation access to over 2.7 billion people globally in low income areas. In urban areas of Ghana and the Philippines, about 85% and 98% of households utilize on-site treatment facilities, respectively . Also, i2) that can be used for cooking or power generation. Anaerobic digestion's cost effectiveness makes it a very attractive treatment for resource-scarce areas. While AD typically does not reduce pathogen to safe levels, a field study conducted by Forbis-Stokes et al. reported that anaerobic digestion of minimally diluted human waste removed about 85–89% of the chemical oxygen demand (COD)  which is the topic of this paper.Anaerobic digestion (AD) is the process of treating organic materials biologically in the absence of oxygen and producing biogas. AD has become a very popular, especially for the treatment of high strength wastes, because it yields an effluent that has significant organic matter reduction (60–80%) as well as producing biogas (about 60% methane and 40% COnd (COD) . HoweverCOD L−1 and 500 mgNH3-H L−1 and was able to achieve removal efficiencies of 79% and 90%, for COD and TAN respectively  removal efficiencies of 66 and 45%, respectively  . This stectively  but inleectively . Uemara ectively . For rawondition . Trickliondition . The ope−1 day−1 and has the potential to precipitate out of solution as struvite (MgNH4PO4·6H2O) which can lead to operational issues in conventional wastewater treatment plants  and urine (619 mg P mL−1) up with efficiencies (based on PO4-P) of 96.7–99.8%  of 25 days. The urine, feces, and water were blended together with an industrial submersible blender and poured into the digester inlet. The digester was an unmixed reactor with loading rates of 1.73 kgCOD m−3 day−1, and 0.80 kgN m−3 day−1. It was operated at room temperature (20 ± 2 °C).A lab-scale anaerobic digester was built to simulate field digesters utilized in on-site sanitation treatment systems. The digester had an 80 L capacity and was fed a mixture of urine, feces and water (6.8:2.7:12.8 vol.) at a rate of 22.4 L weekIn an effort to best simulate an actual field sanitation system, real feces and urine were used to create the influent stream. Dog feces were used instead of human feces, due to ease of collection and availability. The feces were collected periodically from a local dog boarding center and stored at 4 °C until use. Fresh human urine was collected in a portable urinal with integrated storage placed in our laboratory's men's restroom. Both dog feces and urine were stored for no longer than 4 weeks prior to be fed to the anaerobic digester. Tap water was used to simulate minimal flush water. The digester effluent was deemed representative for AD-based on-site sanitation systems in less developed countries.COD L−1 and 2300 mgN L−1. Thus, to operate the filter at loading rates similar to field designs, the anaerobic digestion effluent was further adjusted by slight dilution with tap water and more human urine to achieve an average of about 4500 gCOD L−1 and 3000 gN L−1 influent for each of the trickling filters. Adjusting the digestor effluent resulted in concentrations around 4560 mgCOD L−1 and 2987 mgN L−1, with most (96%) of the nitrogen being in the ammonium form.After initial start-up and operation of the anaerobic digester, the digester effluent was in average 16,100 mg2.2Each filter was filled with 4.86 L of medium. The four trickling filter media types were biochar (BIO), GAC (GAC), coconut husks (COC), and zeolite (ZEO). The four downflow submerged denitrification filter media types were bamboo (BAM), coconut husks (COC), rice husks (RIC), and sunflower seeds (SUN). A wire mesh was placed horizontally between the 10.2 cm ID PVC pipe and the PVC end cap to hold the media up. Media was poured into each filter and compacted every 10 cm.BuyActivatedCharcoal.com. The GAC for Phase 1–3 was derived from coconut shells and also sourced from BuyActivatedCharcoal.com. Coconut husks were sourced from US Orchid Supplies from Oxnard, California. David brand original roasted and salted sunflower seeds were bought at a local grocery store and used for both trickling and submerged filters. Clinoptilolite zeolite was sourced from Pentair Aquatic Eco-Systems. Bamboo (Phyllostachys edulis) was sourced in North Carolina and was shredded to create bamboo chips. The rice husks were sourced from Peaceful Valley Farm & Garden Supply in Grass Valley, California.The biochar for Phase 1 and Phase 2 was made from pine and sourced from Biomass Controls, LLC . The biochar for Phase 3 was Charcoal Green Cocochar biochar made from coconut shell and sourced from 2.3−1 to wash out suspended particles and highly soluble salts. Each trickling filter and submerged filter was inoculated once with 1 L activated sludge and 1 L anaerobic digester effluent from a local wastewater treatment plant, fed over the course of 6 h. The inoculum was fed into the inlet port and the mid-point of each trickling and submerged filter.Before inoculation, tap water was pumped through the DN filters for 3 days at 10 L day2.4COD L−1 and 3000 mgTAN L−1, respectively. Experiments were conducted in a system of four different trickling filters followed each in series by submerged filters , coconut husks (COC), rice husks (RIC), and sunflower seeds (SUN). The downflow denitrification filters were similarly constructed except that the media was submerged, and there was no aeration. To keep the beds submerged, each filter's effluent line was routed upwards to an overflow fitted with an air gap, thus ensuring a constant liquid level in the submerged filters. The tops of each filter bed were also covered with a mesh to constrain floating media.−1. The daily feed pattern consisted of multiple 5-minute intervals with a pump timer at various times throughout the day  to simulate peak flow times. All systems were kept at room temperature (20 ± 2 °C).The anaerobic digester effluent (adjusted by slight dilution) was pumped with peristaltic pumps into the top of each trickling filter at a rate corresponding to a volume of 0.8 L day2.5−1. This was determined based on the average oxygen demand of the influent COD and TN concentrations , this aeration would theoretically be exactly stoichiometric to completely oxidize the contaminants. No changes were made to the experimental conditions of the denitrification filters during Phase 2. The results reflect denitrification filter performance as a result of changes in experimental conditions of the preceeding nitrification filters. After 17 total weeks, the BIO and GAC nitrification filters began to clog and were exchanged for identical but larger-sized fresh media. These experimental changes for Phase 3 were only applicable to the recently changed BIO and GAC media filters. Likewise, with Phase 2, the Phase results of the denitrification filters reflect their performance as a result of changes in experimental conditions of the preceeding nitrification filters , nitrite (NO2-N), nitrate (NO3-N), COD and reactive phosphate (PO4-P) were measured weekly using Hach kits . Dissolved oxygen (DO) and pH were measured using dedicated probes . Turbidity was measured using EPA Method 180.1 , all of which were performed weekly.Starting on Day 0, samples were taken from the outlet of each trickling filters and from the outlet and mid-depth locations of the submerged filters. Total nitrogen (TN), total ammonia nitrogen .33.14+ thus fit to be converted by nitrification to NO2− and NO3− in the trickling filters so that, if desired, these species could subsequently be converted to N2 gas in the submerged anaerobic filters. During Phase 1, the ZEO medium filter had the highest removal of TAN at 69% compared to 25–50% in the other filters  performed the best with 21% COD removal, 30% TAN removal, and 71% PO−1 which is less than half of the COD concentration entering during Phase 1 at 2818 mg L−1, as seen in DO L−1, 0.83–0.89 mgDO L−1, 0.84–0.92 mgDO L−1, respectively for all three operational Phases. The SUN medium filter yield more favorable anoxic conditions and averaged 0.10–0.12 mgDO L−1, which could also explain higher denitrification rates. Furthermore, in Phase 2, the submerged denitrification filters removed significantly higher COD than in Phase 1. This result could also be indicative of the reduced COD loading rate being more favorable to the denitrification process under the designed experimental conditions.Each filter exhibitted increased COD efficiencies in Phase 2 with 32, 26, 23, and −75%, respectively. This indicates that less organics were being leached or more organics were being biologically consumed. Also, for Phase 2, the average COD concentration entering the denitrifying filters was 1277 mg L4-P, respectively, while the SUN medium filter removed 38%. Shifting from Phase 1 to Phase 2, COD removal efficiency increased in all filters and simultaneously became less efficient at removing PO4-P, with exception of the SUN medium filter. This was most apparent in the COC medium filter as performance decreased to 51% removal efficiency. This could further support that the primary removal mechanism of PO4-P is adsorption. The RIC filter during Phase 1 had a COD removal efficiency of −21% and a TN removal efficiency of 30%. For Phase 2 and Phase 3 it had COD removal efficiencies of 23% and 40%, respectively, and TN removal efficiencies of 9% and 13%, respectively.BAM and RIC media filters achieved 66% and 65% removal of PO3-N removal (28–44%) and NO2-N removal (50–56%) by denitrification. Interesting though, these three filters also yielded NH3 removal at 11–21%. This suggests that the submerged denitrification filters are continuing to adsorb NH3. However, throughout each Phase, the RIC filter had similar NO2-N removal efficiencies (54–61%). From Phase 1 to Phase 2, the RIC medium filter also yielded similar NO3-N removal efficiencies at 44% and 42%, respectively, yet increased COD removal efficiencies from −21% to 23%, respectively. In Phase 3, RIC filter's NO3-N efficiency dropped to −23%. The SUN filter, for each Phase, was able to remove almost all the NO2-N and NO3-N in the influent between 96 and 99%.The BAM, COC, and RIC filters for Phase 1 had the highest TN removal efficiency of 30% , Table 53.3After about 35 days of operation, it was observed that a white crystal precipitate was forming in the denitrification SUN medium filter effluent tubing. Samples were extracted from the submerged SUN filter outlet port and dried in a furnace at 550 °C for 1 h. X-ray diffraction analysis was performed to characterize the crystalline solid. Analysis yielded presence of ammonium magnesium phosphate hydrate and ammonium magnesium hydrogen phosphate hydrate. Ammonium magnesium phosphate, also known as struvite, has been demonstrated to be a slow-release nutrient fertilizer that is useful for agricultural purposes.44.1−1 to 0.1–1.9 mg L−1. In addition to the simultaneous clogging of the BIO and GAC media filters, it was determined that the natural air flow into the filter was not enough for all the fast-growing heterotrophic bacteria and autotrophic nitrifying bacteria. It was then decided to pump air into the filters (i.e. Phase 2).The first phase of the experiments relied on aeration by natural convection. However, after about 12 weeks, oxygen became limiting and the DO concentration of the filter effluent started to decrease from 3.6–4.8 mg LInitially, in Phase 1, the BIO and GAC filter media were used. At the beginning of Phase 1, the influent was able to trickle all the way through the filter. However, after about 14 weeks, the filters started to clog at the top. Because of the small effective media diameter of 1.5 mm and 0.3 mm, the biochar and GAC also served as physical filter for suspended solids in the influent. To declog the filters, a layer of media and influent solids, about 1 cm thick, was removed from the top of the filter, and operation was resumed. Clogs then soon started to be a reoccurring issue, about every week. Thus, it was decided to replace the BIO and GAC filter media with larger effective sized diameter media of 4.0 mm and 2.4 mm, respectively, such that the filters would serve less as a physical filter and more as a biological filter.4.24+, PO43− and Mg2+ are combined, struvite is formed. Since most of the nitrogen species in the trickling filter is in the form of NH4+, it was thought that the trickling filter would be a good place for struvite formation.It was initially thought that using sunflower seeds as a trickling filter media would be good to produce struvite because of their high magnesium content. Sunflower seeds are a cheap, sustainable crop that can be grown in many places around the world. Interestingly, sunflower seeds have some of the highest magnesium concentrations of all foods. When a 1:1:1 ratio of NHCOD L−1 and 13,080 mgCOD L−1, respectively in Phase 3. After about 4 weeks of operation, flies started to appear in the laboratory and the sunflower seed trickling filter sample port became clogged with maggots. Flies were not present in other filters. The sunflower seed trickling filter was decommissioned and replaced with coconut husks (same as in denitrification filters). Although, the temporary sunflower seed filters promoted some nitrification, using sunflower seeds as trickling filter medium would not be practical for field use because of the excessive leaching of organics. This experience suggests that other media which contain readily biodegradable materials should not be used.After the start of the experiment, a lot of the organic matter from the sunflower seeds (seed and shell) was leaching into the treated wastewater, particularly in the later phases. This resulted in higher average COD effluent concentration than influent concentration, 2818 mg2-N and NO3-N loading. With removal rates of 0.037–0.068 kgNO2 m−3 day−1 and 0.041–0.091 kgNO3 m−3 day−1, the SUN medium denitrification filters removed 99% and 96–99% of influent NO2-N and NO3-N, respectively.Similarly, the readily biodegradable materials were thought to be a cause of concern for the respective submerged denitrification filter. The sunflower seed and rice husk submerged denitrification filter yielded negative COD removal, as the rate of organics leaching into the wastewater was greater than the biological organic consumption rate, though the sunflower seed submerged filter was the only one to precipitate ammonium magnesium phosphate. It may be more practically feasible to combine sunflower seeds with other media type to achieve both denitrification and organic matter removal. However, in the context that high organic matter in effluent is less of an issue, sunflower seed medium submerged denitrification filters could serve as a very effective option to remove almost all NO4.34-P up to 84%, 69%, and 89%, respectively, partly meeting the treatment requirements (80% load reduction for P and 70% for N) of a newly published ISO standard (30500) for non-sewered sanitation systems. However, the effluent is not polished enough to meet discharge or reuse standards completely, thus optimization of post-treatment processes is necessary to meet standards.Experimental data from laboratory operation have demonstrated combined removal of COD, TN, and PO4-P removal rates of 0.252 kgTAN m−3 day−1 and 0.15 kgP m−3 day−1, respectively. This is high compared to the 0.102 kgTAN m−3 day−1 removal rate achieved by TAN m−3 day−1) compared to the TAN m−3 day−1). The 196% increase in TAN loading rate (because of higher TAN concentrations) yielded a 147% increase in removal rate indicating positive dependency of nitrification rates with the TAN concentration at the conditions of the experiment. From the COC medium denitrification filter during Phase 1, the nitrification-denitrification filter system was able to achieve a PO4-P removal efficiency of 89%. The ZEO medium filter also yielded 58% COD removal at 0.35 kgCOD m−3 day−1. Based on the observed removal rates, an actual sanitation system for a family of 5 would need the greater of 160, 248, and 97-liter filter packed with ZEO to remove close to 100% of COD, TN, and PO4-P, respectively from their anaerobically digested human wastes. Thus sizing (248 liter) would be determined by TN removal.In a practical application where nutrient removal is prioritized, the ZEO medium filter with natural aeration (Phase 1) yielded the highest overall nutrient removal performance with TAN and PO4-P removal rates of 0.22 kgTAN m−3 day−1 and 0.094 kgP m−3 day−1, respectively.In a context where organic contaminant removal is prioritized, it is recommended that the GAC filter with forced aeration (Phase 2) be used as it yielded the highest COD removal at 84%. The cost implications of such aeration, while moderate, would need to be considered. The GAC filter also supported relatively high TAN and POThroughout the duration of the study, the COC and ZEO filter media did not have to be changed. However, the BIO and GAC filter media began to experience clogging after around 12 weeks of operation. If BIO or GAC media trickling filters are to be operated in field conditions, it is recommended that larger diameter media be used to avoid quarterly media replacement.5The fertilizer-generating anaerobic digestion post-treatment filter system simultaneously addresses the global lack of access to improved sanitation and could potentially expand the capacity for small-scale agricultural development when combined with on-site sanitation technologies like the Anaerobic Pasteurization Latrine (ADPL) . PilotedWe wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.


Antibodies are essential mediators of immunological defense mechanisms, are clinically used as therapeutic agents, but are also functionally involved in various immune-mediated disorders. Whereas IgG antibodies accomplish some of their biological tasks autonomously, many functions depend on their binding to activating and inhibitory Fcγ receptors (FcγR). From a qualitative point of view expression patterns of FcγR on immunologically relevant cell types are well-characterized both for mice and humans. Surprisingly, however, there is only quite limited information available on actual quantities of FcγR expressed by the different leukocyte populations. In this study we provide a comprehensive data set assessing quantitatively how many individual human and mouse FcγRs are expressed on B cells, NK cells, eosinophils, neutrophils, basophils and both classical, and non-classical monocytes under steady state conditions. Moreover, among human donors we found two groups with different expression levels of the inhibitory FcγRIIb on monocytes which appears to correlate with haplotypes of the activating FcγRIIIa. Antibodies of the immunoglobulin G (IgG) isotype are essential mediators of immunological defense mechanisms. Whereas they carry out some of their biological functions autonomously, such as blocking of cell surface receptors, most of their functions depend on the binding of the antibody Fc-domain to specific receptors, the Fc gamma receptors (FcγR). Whereas binding of immune complexes (IC) is possible to all FcγR including the low/medium receptors FcγRIIa and FcγRIIb, FcγRIIIa and FcγRIIIb in man and FcγRIIb, FcγRIII and FcγRIV in mice, monomeric IgG molecules can only bind to human or murine high affinity receptor FcγRI .The repertoire of murine and human FcγR includes the inhibitory FcγRIIb, which contains an intracellular immunomodulatory tyrosine based inhibitory motif (ITIM) whereas most other FcγR carry an activating ITAM motif. These motifs are either located in the same polypeptide chain as the ligand binding domain (FcγRIIb and FcγRIIIb) or on an accessory Fc receptor gamma chain. One exception is the human FcγRIIIb which contains no transmembrane or intracellular polypeptide domain, but is rather linked to the cell membrane by a GPI anchor .Immune cell populations are characterized by a typical expression pattern of FcγR. Whereas most of them express both activating and inhibitory FcγR, B cells only express the inhibitory FcγRIIb, which regulates the activity of the B cell receptor. Natural Killer (NK) cells on the other side selectively express the activating FcγRIII. Thus, from a qualitative point of view expression patterns of FcγR on different immune cell subsets are well-characterized both, for mice and man –3, 5–12.For many cell types, their functional response to external signals is accompanied by pronounced up- or down-regulation of various surface receptors. This clearly shows that the amount of any given surface receptor on a cell is an important characteristic for its physiological functionality. The outcome of immune complex binding to a cell is determined by the sum of activating and inhibitory signals triggered through the respective activating and inhibitory FcγRs. One factor influencing these signals is the affinity of the respective IgG isotype to the distinct FcγRs present on the cell surface. This led to the development of the concept of the so called A/I ratio as a prediction of the outcome of binding of IgG molecules of a given isotype based on its affinities to the inhibitory and activating receptors, respectively –15. BaseWhereas FcγR expression is rather well-described qualitatively, there is only quite limited information available on actual numbers of FcγR expressed by the different leukocyte populations –35. To oFlow cytometry is a common method for the analysis of cell surface receptors. Qualitative comparison of the relative expression of a given cell surface receptor on different cell populations based on the fluorescence intensities mediated by binding of fluorochrome-labeled antibodies specific for this receptor is rather straightforward. In contrast, quantitative comparison of different receptors on the same or different cell populations by simple comparison of fluorescence intensities is not possible since there are inherent variations between the different anti-receptor antibody conjugates with respect to their specific fluorescence . This is obvious when conjugates with different fluorochromes are used. However, even antibodies conjugated with the same fluorochrome may differ in the stoichiometry of fluorophores per antibody molecule. This variability can be partially reduced by using conjugates with very large fluorophores like R-Phycoerythrin (PE), where usually not more than one fluorochrome can be linked to each antibody molecule . In spitantibody-binding capacity for respective anti-FcγR antibodies) for all activating and inhibitory FcγR on the main leukocyte populations in peripheral blood of C57BL/6 and FcγR knockout mice and in healthy human volunteers under steady state conditions. This includes Natural Killer (NK) cells, B cells, T cells, neutrophils, eosinophils and basophils as well as classical and non-classical monocytes. These monocyte subsets have comparable population size in murine blood. In contrast, in humans the classical monocytes represent the vast majority of monocytes whereas non-classical monocytes represent only a small fraction of the monocyte population but nonetheless exert important biological functions. In both species these subsets have differential FcγR repertoires and, thus, specific effector functions upon engagement with immune complexes  and  does not affect FcγRIIb expression on B cells  detectable in individuals with low affinity FcγRIIa/FcγRIIIa haplotypes. Following this line of argument, the modulation of inhibitory receptor expression by activating receptor haplotypes may well be cell type dependent, since this is relevant mainly for cell types that co-express activating and inhibitory receptors, such as monocytes. Accordingly, we found no obvious association of FcγRIIb expression with the activating receptor haplotypes. For the FcγRIIa B cells .How genetic regulation of FcγRIIb expression by activating receptor haplotypes might be achieved on a cellular and molecular level is not yet clear. However, one might envision that the association of FcγRIIb expression and the activating Fcγ-receptor affinity haplotype could be via a promoter/enhancer SNP that modulates expression of FcγRIIb in linkage disequilibrium with coding SNPs of activating Fc receptors as has been suggested by Roederer et al. .According to our results, FcγRIIIb seems to be expressed at exceedingly high levels by human neutrophils. This high amount of FcγRIIIb may be important to fulfill its biological function in immune-complex induced neutrophil activation. It has been suggested to be critical for tethering immune complexes to neutrophils . It may In summary, we present a comprehensive and comparative numerical quantification of Fcγ receptors on the main immune cell subsets in humans and the most common laboratory mouse strain C57BL/6J. These numerical data may enable improved stoichiometric considerations on A/I ratios regarding activating and inhibitory receptors as well as improved modeling of antibody-mediated immunological processes. In addition, we emphasize that conclusions drawn from knockout animal models have to be carefully evaluated in order to avoid potential misinterpretations due to compensatory modulation of other receptors.10.6084/m9.figshare.11604201.The datasets generated for this study can be found in the Figshare repository: doi: The studies involving human participants were reviewed and approved by Ethics Commission of the University of Erlangen-Nürnberg. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Government of Lower Franconia.MB and CK performed experiments with equal contribution and analyzed data. MB and FN supervised experiments, interpreted data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.