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Neurons communicate via synapses—specialized structures that consist of a presynaptic terminal of one neuron and a postsynaptic terminal of another. As knowledge is emerging that mutations in molecules that regulate synaptic function underpin many neurological disorders, it is crucial to elucidate the molecular mechanisms regulating synaptic function to understand synaptic strength, plasticity, modulation, and pathology, which ultimately impact neuronal circuit output and behavior. The presynaptic calyx of Held is a large glutamatergic presynaptic terminal in the auditory brainstem, which due to its accessibility and the possibility to selectively perform molecular perturbations on it, is an ideal model to study the role of presynaptic proteins in regulating synaptic function. In this protocol, we describe the use of confocal imaging and three-dimensional reconstruction of the calyx of Held to assess alterations in gross morphology following molecular perturbation. Using viral-vector delivery to perform molecular perturbations at distinct developmental time points, we provide a fast and cost-effective method to investigate how presynaptic proteins regulate gross morphology such as surface area and synapse volume throughout the lifetime of a neuronal circuit.

Neurons communicate via synapses—specialized structures that consist of a presynaptic terminal of one neuron and a postsynaptic terminal of another. As knowledge is emerging that mutations in molecules that regulate synaptic function underpin many neurological disorders, it is crucial to elucidate the molecular mechanisms regulating synaptic function to understand synaptic strength, plasticity, modulation, and pathology, which ultimately impact neuronal circuit output and behavior. The presynaptic calyx of Held is a large glutamatergic presynaptic terminal in the auditory brainstem, which due to its accessibility and the possibility to selectively perform molecular perturbations on it, is an ideal model to study the role of presynaptic proteins in regulating synaptic function. In this protocol, we describe the use of confocal imaging and three-dimensional reconstruction of the calyx of Held to assess alterations in gross morphology following molecular perturbation. Using viral-vector delivery to perform molecular perturbations at distinct developmental time points, we provide a fast and cost-effective method to investigate how presynaptic proteins regulate gross morphology such as surface area and synapse volume throughout the lifetime of a neuronal circuit.

Neurons communicate via synapses—specialized structures that consist of a presynaptic terminal of one neuron and a postsynaptic terminal of another. As knowledge is emerging that mutations in molecules that regulate synaptic function underpin many neurological disorders, it is crucial to elucidate the molecular mechanisms regulating synaptic function to understand synaptic strength, plasticity, modulation, and pathology, which ultimately impact neuronal circuit output and behavior. The presynaptic calyx of Held is a large glutamatergic presynaptic terminal in the auditory brainstem, which due to its accessibility and the possibility to selectively perform molecular perturbations on it, is an ideal model to study the role of presynaptic proteins in regulating synaptic function. In this protocol, we describe the use of confocal imaging and three-dimensional reconstruction of the calyx of Held to assess alterations in gross morphology following molecular perturbation. Using viral-vector delivery to perform molecular perturbations at distinct developmental time points, we provide a fast and cost-effective method to investigate how presynaptic proteins regulate gross morphology such as surface area and synapse volume throughout the lifetime of a neuronal circuit.

A surface soil moisture (SM) condition at high spatiotemportal resolutions is required by regional Earth system applications. Here, we mapped daily 1-km SM in the Babao River Basin in the northwest of China during the summers from 2013 to 2015 using a random forest (RF) method by merging SM information retrieved from in situ measurements, optical/thermal remote sensing, and topographical indices. Relative importance analysis was used to determine the optimal predictors for estimating high-resolution SM. A specific RF model (RFVI+sup) was constructed using the optimal predictors including remote sensing albedo, apparent thermal inertia (ATI), normalized difference vegetation index, normalized difference infrared index 5, soil adjusted vegetation index, and topographical indices (aspect and elevation). The RFVI+sup also accounted for missing observations of the thermal index (e.g., ATI) over the mountainous regions. In the comparison between the SM estimates using the new RFVI+sup model and other RF models, the spatial coverage of available estimates increased from 14% to 64% over the study region, the correlation coefficient values were improved to 0.75, the unbiased root-mean-squared difference values decreased to 0.032 m3/m3. Thus, the proposed RF method provided accurate SM estimates with high spatiotemporal resolution over the mountainous regions, by merging multiresource datasets from in situ measurements, remotely-sensed, and topographical indices.

Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1’s roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability.

Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1’s roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability.

Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1’s roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability.

Long-term and high-quality surface soil moisture (SSM) and root-zone soil moisture (RZSM) data is crucial for understanding the land-atmosphere interactions of the Qinghai-Tibet Plateau (QTP). More than 40% of QTP is covered by permafrost, yet few studies have evaluated the accuracy of SSM and RZSM products derived from microwave satellite, land surface models (LSMs) and reanalysis over that region. This study tries to address this gap by evaluating a range of satellite and reanalysis estimates of SSM and RZSM in the thawed soil overlaying permafrost in the QTP, using in-situ measurements from sixteen stations. Here, seven SSM products were evaluated: Soil Moisture Active Passive L3 (SMAP-L3) and L4 (SMAP-L4), Soil Moisture and Ocean Salinity in version IC (SMOS-IC), Land Parameter Retrieval Model (LPRM) Advanced Microwave Scanning Radiometer 2 (AMSR2), European Space Agency Climate Change Initiative (ESA CCI), Advanced Scatterometer (ASCAT), and the fifth generation of the land component of the European Centre for Medium-Range Weather Forecasts atmospheric reanalysis (ERA5-Land). We also evaluated three RZSM products from SMAP-L4, ERA5-Land, and the Noah land surface model driven by Global Land Data Assimilation System (GLDAS-Noah). The assessment was conducted using five statistical metrics, i.e. Pearson correlation coefficient (R), bias, slope, Root Mean Square Error (RMSE), and unbiased RMSE (ubRMSE) between SSM or RZSM products and in-situ measurements. Our results showed that the ESA CCI, SMAP-L4 and SMOS-IC SSM products outperformed the other SSM products, indicated by higher correlation coefficients (R) (with a median R value of 0.63, 0.44 and 0.57, respectively) and lower ubRMSE (with a median ubRMSE value of 0.05, 0.04 and 0.07 m3/m3, respectively). Yet, SSM overestimation was found for all SSM products. This could be partly attributed to ancillary data used in the retrieval (e.g. overestimation of land surface temperature for SMAP-L3) and to the fact that

The vegetation optical depth (VOD), a vegetation index retrieved from passive or active microwave remote sensing systems, is related to the intensity of microwave extinction effects within the vegetation canopy layer. This index is only marginally impacted by effects from atmosphere, clouds and sun illumination, and thus increasingly used for ecological applications at large scales. Newly released VOD products show different abilities in monitoring vegetation features, depending on the algorithm used and the satellite frequency. VOD is increasingly sensitive to the upper vegetation layer as the frequency increases (from L-, C- to X-band), offering different capacities to monitor seasonal changes of the leafy and/or woody vegetation components, vegetation water status and aboveground biomass. This study evaluated nine recently developed/reprocessed VOD products from the AMSR2, SMOS and SMAP space-borne instruments for monitoring structural vegetation features related to phenology, height and aboveground biomass.For monitoring the seasonality of green vegetation (herbaceous and woody foliage), we found that X-VOD products, particularly from the LPDR-retrieval algorithm, outperformed the other VOD products in regions that are not densely vegetated, where they showed higher temporal correlation values with optical vegetation indices (VIs). However, LPDR X-VOD time series failed to detect changes in VOD after rainfall events whereas most other VOD products could do so, and overall daily variations are less pronounced in LPDR X-VOD. Results show that the reprocessed VODCA C- and X-VOD have almost comparable performance and VODCA C-VOD correlates better with VIs than other C-VOD products. Low frequency L-VOD, particularly the new version (V2) of SMOS-IC, show a higher temporal correlation with VIs, similar to C-VOD, in medium-densely vegetated biomes such as savannas (R similar to 0.70) than for other short vegetation types. Because the L-VOD indices are more sen

The vegetation optical depth (VOD), a vegetation index retrieved from passive or active microwave remote sensing systems, is related to the intensity of microwave extinction effects within the vegetation canopy layer. This index is only marginally impacted by effects from atmosphere, clouds and sun illumination, and thus increasingly used for ecological applications at large scales. Newly released VOD products show different abilities in monitoring vegetation features, depending on the algorithm used and the satellite frequency. VOD is increasingly sensitive to the upper vegetation layer as the frequency increases (from L-, C- to X-band), offering different capacities to monitor seasonal changes of the leafy and/or woody vegetation components, vegetation water status and aboveground biomass. This study evaluated nine recently developed/reprocessed VOD products from the AMSR2, SMOS and SMAP space-borne instruments for monitoring structural vegetation features related to phenology, height and aboveground biomass.For monitoring the seasonality of green vegetation (herbaceous and woody foliage), we found that X-VOD products, particularly from the LPDR-retrieval algorithm, outperformed the other VOD products in regions that are not densely vegetated, where they showed higher temporal correlation values with optical vegetation indices (VIs). However, LPDR X-VOD time series failed to detect changes in VOD after rainfall events whereas most other VOD products could do so, and overall daily variations are less pronounced in LPDR X-VOD. Results show that the reprocessed VODCA C- and X-VOD have almost comparable performance and VODCA C-VOD correlates better with VIs than other C-VOD products. Low frequency L-VOD, particularly the new version (V2) of SMOS-IC, show a higher temporal correlation with VIs, similar to C-VOD, in medium-densely vegetated biomes such as savannas (R similar to 0.70) than for other short vegetation types. Because the L-VOD indices are more sen

The vegetation optical depth (VOD), a vegetation index retrieved from passive or active microwave remote sensing systems, is related to the intensity of microwave extinction effects within the vegetation canopy layer. This index is only marginally impacted by effects from atmosphere, clouds and sun illumination, and thus increasingly used for ecological applications at large scales. Newly released VOD products show different abilities in monitoring vegetation features, depending on the algorithm used and the satellite frequency. VOD is increasingly sensitive to the upper vegetation layer as the frequency increases (from L-, C- to X-band), offering different capacities to monitor seasonal changes of the leafy and/or woody vegetation components, vegetation water status and aboveground biomass. This study evaluated nine recently developed/reprocessed VOD products from the AMSR2, SMOS and SMAP space-borne instruments for monitoring structural vegetation features related to phenology, height and aboveground biomass. For monitoring the seasonality of green vegetation (herbaceous and woody foliage), we found that X-VOD products, particularly from the LPDR-retrieval algorithm, outperformed the other VOD products in regions that are not densely vegetated, where they showed higher temporal correlation values with optical vegetation indices (VIs). However, LPDR X-VOD time series failed to detect changes in VOD after rainfall events whereas most other VOD products could do so, and overall daily variations are less pronounced in LPDR X-VOD. Results show that the reprocessed VODCA C- and X-VOD have almost comparable performance and VODCA C-VOD correlates better with VIs than other C-VOD products. Low frequency L-VOD, particularly the new version (V2) of SMOS-IC, show a higher temporal correlation with VIs, similar to C-VOD, in medium-densely vegetated biomes such as savannas (R ~ 0.70) than for other short vegetation types. Because the L-VOD indices are more sensitive to the non

Changes in terrestrial tropical carbon stocks have an important role in the global carbon budget. However, current observational tools do not allow accurate and large-scale monitoring of the spatial distribution and dynamics of carbon stocks1. Here, we used low-frequency L-band passive microwave observations to compute a direct and spatially explicit quantification of annual aboveground carbon (AGC) fluxes and show that the tropical net AGC budget was approximately in balance during 2010 to 2017, the net budget being composed of gross losses of −2.86 PgC yr−1 offset by gross gains of −2.97 PgC yr−1 between continents. Large interannual and spatial fluctuations of tropical AGC were quantified during the wet 2011 La Niña year and throughout the extreme dry and warm 2015–2016 El Niño episode. These interannual fluctuations, controlled predominantly by semiarid biomes, were shown to be closely related to independent global atmospheric CO2 growth-rate anomalies (Pearson’s r = 0.86), highlighting the pivotal role of tropical AGC in the global carbon budget.

Plant water storage is fundamental to the functioning of terrestrial ecosystems by participating in plant metabolism, nutrient and sugar transport, and maintenance of the integrity of the hydraulic system of the plant. However, a global view of the size and dynamics of the water pools stored in plant tissues is still lacking. Here, we report global patterns of seasonal variations in ecosystem-scale plant water storage and their relationship with leaf phenology, based on space-borne measurements of L-band vegetation optical depth. We find that seasonal variations in plant water storage are highly synchronous with leaf phenology for the boreal and temperate forests, but asynchronous for the tropical woodlands, where the seasonal development of plant water storage lags behind leaf area by up to 180 days. Contrasting patterns of the time lag between plant water storage and terrestrial groundwater storage are also evident in these ecosystems. A comparison of the water cycle components in seasonally dry tropical woodlands highlights the buffering effect of plant water storage on the seasonal dynamics of water supply and demand. Our results offer insights into ecosystem-scale plant water relations globally and provide a basis for an improved parameterization of eco-hydrological and Earth system models.

The vegetation optical depth (VOD) measured at microwave frequencies is related to the vegetation water content and provides information complementary to visible/infra-red vegetation indices. This study is devoted to the characterisation of a new VOD data set obtained from SMOS (Soil Moisture and Ocean Salinity) satellite observations at L-band (1.4 GHz). Three different SMOS L-band VOD (L-VOD) data sets (SMOS Level 2, Level 3 and SMOS-IC) were compared with data sets on tree height, visible/infra-red indexes (NDVI, EVI), cumulated precipitation, and above ground biomass (AGB) for the African continent. For all relationships, SMOS-IC showed the lowest dispersion and highest correlation. Overall, we found a strong (R > 0.85) correlation with no clear sign of saturation between L-VOD and four AGB data sets. The relationship linking L-VOD to tree height (R = 0.87) and Baccini's AGB (R = 0.94) was strong and linear. The relationships between L-VOD and three other AGB data sets were linear per land cover class, but with a changing slope depending on the land cover type. For low vegetation classes, the annual mean of L-VOD spans a range from 0 to 0.7 and it is linearly correlation with the amount of the average annual precipitations. SMOS L-VOD showed a higher sensitivity to AGB as compared to NDVI and K/X/C-VOD (VOD measured, respectively, at 19, 10.7, and 6.9 GHz). The results showed that although the spatial resolution of L-VOD is coarse (~ 40 km), the high temporal frequency and sensitivity to AGB makes SMOS L-VOD a very promising index for large scale monitoring of the vegetation status, in particular biomass.

Tropical aboveground carbon changes during 2010-2016 were estimated by a newly developed vegetation optical depth (VOD) product retrieved from the low-frequency L-band (1.4 GHz) passive microwave observations from the Soil Moisture and Ocean salinity (SMOS) satellite. The aboveground carbon changes estimated by VOD in the tropical region during 2010-2016 indicate the tropical region acts as a net carbon source of 111 Tg C yr-1 during 2010-2016. The declines in tropical aboveground carbon were found mainly in eastern America, African drylands and Indonesia.

The vegetation optical depth (VOD) measured at microwave frequencies is related to the vegetation water content and provides information complementary to visible/infra-red vegetation indices. This study is devoted to the characterisation of a new VOD data set obtained from SMOS (Soil Moisture and Ocean Salinity) satellite observations at L-band (1.4 GHz). Three different SMOS L-band VOD (L-VOD) data sets (SMOS Level 2, Level 3 and SMOS-IC) were compared with data sets on tree height, visible/infra-red indexes (NDVI, EVI), cumulated precipitation, and above ground biomass (AGB) for the African continent. For all relationships, SMOS-IC showed the lowest dispersion and highest correlation. Overall, we found a strong (R > 0.85) correlation with no clear sign of saturation between L-VOD and four AGB data sets. The relationship linking L-VOD to tree height (R = 0.87) and Baccini's AGB (R = 0.94) was strong and linear. The relationships between L-VOD and three other AGB data sets were linear per land cover class, but with a changing slope depending on the land cover type. For low vegetation classes, the annual mean of L-VOD spans a range from 0 to 0.7 and it is linearly correlation with the amount of the average annual precipitations. SMOS L-VOD showed a higher sensitivity to AGB as compared to NDVI and K/X/C-VOD (VOD measured, respectively, at 19, 10.7, and 6.9 GHz). The results showed that although the spatial resolution of L-VOD is coarse (~ 40 km), the high temporal frequency and sensitivity to AGB makes SMOS L-VOD a very promising index for large scale monitoring of the vegetation status, in particular biomass.

The objective of this paper is to present the multi-orbit (MO) surface soil moisture (SM) and angle-binned brightness temperature (TB) products for the SMOS (Soil Moisture and Ocean Salinity) mission based on a new multi-orbit algorithm. The Level 3 algorithm at CATDS (Centre Aval de Traitement des Donnees SMOS) makes use of MO retrieval to enhance the robustness and quality of SM retrievals. The motivation of the approach is to make use of the longer temporal autocorrelation length of the vegetation optical depth (VOD) compared to the corresponding SM autocorrelation in order to enhance the retrievals when an acquisition occurs at the border of the swath. The retrieval algorithm is implemented in a unique operational processor delivering multiple parameters (e.g. SM and VOD) using multi-angular dual-polarisation TB from MO. A subsidiary angle-binned TB product is provided. In this study the Level 3 TB V310 product is showcased and compared to SMAP (Soil Moisture Active Passive) TB. The Level 3 SM V300 product is compared to the single-orbit (SO) retrievals from the Level 2 SM processor from ESA with aligned configuration. The advantages and drawbacks of the Level 3 SM product (L3SM) are discussed. The comparison is done on a global scale between the two datasets and on the local scale with respect to in situ data from AMMA-CATCH and USDA ARS Watershed networks. The results obtained from the global analysis show that the MO implementation enhances the number of retrievals: up to 9% over certain areas. The comparison with the in situ data shows that the increase in the number of retrievals does not come with a decrease in quality, but rather at the expense of an increased time lag in product availability from 6 h to 3.5 days, which can be a limiting factor for applications like flood forecast but reasonable for drought monitoring and climate change studies. The SMOS L3 soil moisture and L3 brightness temperature products are delivered using an open licence and free o

The objective of this paper is to present the multi-orbit (MO) surface soil moisture (SM) and angle-binned brightness temperature (TB) products for the SMOS (Soil Moisture and Ocean Salinity) mission based on a new multi-orbit algorithm. The Level 3 algorithm at CATDS (Centre Aval de Traitement des Donnees SMOS) makes use of MO retrieval to enhance the robustness and quality of SM retrievals. The motivation of the approach is to make use of the longer temporal autocorrelation length of the vegetation optical depth (VOD) compared to the corresponding SM autocorrelation in order to enhance the retrievals when an acquisition occurs at the border of the swath. The retrieval algorithm is implemented in a unique operational processor delivering multiple parameters (e.g. SM and VOD) using multi-angular dual-polarisation TB from MO. A subsidiary angle-binned TB product is provided. In this study the Level 3 TB V310 product is showcased and compared to SMAP (Soil Moisture Active Passive) TB. The Level 3 SM V300 product is compared to the single-orbit (SO) retrievals from the Level 2 SM processor from ESA with aligned configuration. The advantages and drawbacks of the Level 3 SM product (L3SM) are discussed. The comparison is done on a global scale between the two datasets and on the local scale with respect to in situ data from AMMA-CATCH and USDA ARS Watershed networks. The results obtained from the global analysis show that the MO implementation enhances the number of retrievals: up to 9% over certain areas. The comparison with the in situ data shows that the increase in the number of retrievals does not come with a decrease in quality, but rather at the expense of an increased time lag in product availability from 6 h to 3.5 days, which can be a limiting factor for applications like flood forecast but reasonable for drought monitoring and climate change studies. The SMOS L3 soil moisture and L3 brightness temperature products are delivered using an open licence and free o

The objective of this paper is to present the multi-orbit (MO) surface soil moisture (SM) and angle-binned brightness temperature (TB) products for the SMOS (Soil Moisture and Ocean Salinity) mission based on a new multi-orbit algorithm. The Level 3 algorithm at CATDS (Centre Aval de Traitement des Donnees SMOS) makes use of MO retrieval to enhance the robustness and quality of SM retrievals. The motivation of the approach is to make use of the longer temporal autocorrelation length of the vegetation optical depth (VOD) compared to the corresponding SM autocorrelation in order to enhance the retrievals when an acquisition occurs at the border of the swath. The retrieval algorithm is implemented in a unique operational processor delivering multiple parameters (e.g. SM and VOD) using multi-angular dual-polarisation TB from MO. A subsidiary angle-binned TB product is provided. In this study the Level 3 TB V310 product is showcased and compared to SMAP (Soil Moisture Active Passive) TB. The Level 3 SM V300 product is compared to the single-orbit (SO) retrievals from the Level 2 SM processor from ESA with aligned configuration. The advantages and drawbacks of the Level 3 SM product (L3SM) are discussed. The comparison is done on a global scale between the two datasets and on the local scale with respect to in situ data from AMMA-CATCH and USDA ARS Watershed networks. The results obtained from the global analysis show that the MO implementation enhances the number of retrievals: up to 9% over certain areas. The comparison with the in situ data shows that the increase in the number of retrievals does not come with a decrease in quality, but rather at the expense of an increased time lag in product availability from 6 h to 3.5 days, which can be a limiting factor for applications like flood forecast but reasonable for drought monitoring and climate change studies. The SMOS L3 soil moisture and L3 brightness temperature products are delivered using an open licence and free o