The real time assessment of the distances between the bottom of the drilled cavities and the top of the pulp chamber was performed using an own assembled OCT system. The evaluation of the remaining dentin thickness (RDT) allowed for the positioning of the drilling tools in the cavities in relation to the pulp horns. Estimations of the safe and of the critical RDT were made; for the latter, the opening of the pulp chamber becomes unavoidable. Also, by following the fractures that can occur when the extent of the decay is too large, the dentist can decide upon the right therapy to follow, endodontic or conventional filling. The study demonstrates the usefulness of OCT imaging in guiding such evaluations during dental procedures.A routine cavity preparation of a tooth may lead to opening the pulp chamber. The present study evaluates quantitatively, in real time, for the first time to the best of our knowledge, the drilled cavities during dental procedures. An established noninvasive imaging technique, Optical Coherence Tomography (OCT), is used. The main scope is to prevent accidental openings of the dental pulp chamber. Six teeth with dental cavities have been used in this  The pulp chamber is the cavity situated in the central portion of the tooth , 2, contIn frontal teeth, the pulp chamber is partially located in the crown, whilst in posterior teeth, in the cervical part of the root. Each chamber has a roof at its incisal or occlusal margin; this roof presents projections that are called pulp horns. Normaly one pulp horn is found in each cusped tooth—molars, premolars, and canines. However, in young incisors three pulp horns can be found, while one of the types of maxillary lateral incisors has only one pulp horn. These different morphological aspects, as well as the exact locations of the pulp horns, cannot be determined with conventional procedures such as radiography, due to its low resolution. Radiography is also invasive due to its well-known irradiation issue and it can be applied only before dental procedures and not during them.Cavities/dental caries affect the teeth, therefore their drilling and cleaning, followed by fillings with different direct restorative dental materials is necessary. A major issue of such procedures is that a routine cavity preparation may lead to opening the pulp chamber because of its complex morphology, as mentioned above or due to possible complications of an even simple dental procedure . TherefoThe present work represents an approach to test a noninvasive, real time monitoring method that allows us to avoid such situations and to prevent accidental openings of the pulp chamber during dental procedures. Of course, such openings are sometimes unavoidable, as the depth of a restoration preparation is often based on the extent of the decay and cannot in such cases be controlled by the dentist. However, when the depth of the decay allows, the dentist should be empowered with an instrument to evaluate the RDT. This should allow the dentist to accurately position the drilling tools in the cavities in relation to the pulp horns. In contrast, nowadays the dentist is only able to identify visually—by seeing the blushing of the pulp through the dentin—an RDT that is so small  that an exposure of the pulp chamber already becomes unavoidable. Our study aims to prevent such situation, while also providing an assessment of such an RDT.Training is the next factor in determining the proper depth of cavity preparation. Therefore we will show how the instrument presented here can be also utilized in teaching/demonstrating the impact of cavity preparation in schools, in addition to being put to practical clinical use.In this study, in order to achieve these aims, we evaluate the possibility of using an advanced and well-established biomedical imaging technique, Optical Coherence Tomography (OCT) –10, to i ex vivo the OCT capabilities to be utilized as a noninvasive technique to guide the dentist during dental procedures. Thus, a quantitative assessment of the RDT is performed, during procedures, and estimations are made on the safety and critical RDT. This proves OCT as a valuable tool prior to dental procedures and most important for real time assessment and treatment of caries.To the best of our knowledge, despite the number of OCT studies already performed in dentistry, this is the first time when OCT is applied in the real time monitoring of RDT during caries excavation and cavity preparation. Another scope of this study is to evaluate how OCT can be utilized to investigate the morphology of the dental pulp chamber and to precisely localize the pulp horns. We also demonstrateSix extracted teeth with mesial cavities were used for the investigation. OCT imaging was performed while the dental procedures were applied on each of them, in order to determine the RDT in real time as well as localize the pulp chamber in relationship to the drilled cavity. After completion of the dental procedures, each sample was sectioned in two halves, in order to evaluate the morphology of the pulp chamber at the end of the procedure, to measure directly the final RDT and thus to validate its value obtained using the OCT images—for each tooth.An own assembled Time Domain (TD) OCT system , describ1). The sample arm of the interferometer comprises two microscope objectives (MO1 and MO2) and a flat mirror (FM) to focus the light on the sample , where it interferes with that coming from the reference arm. Polarization controllers (PC1 and PC2) are positioned on each of the interferometer arms. Both output fibres from (DC2) are connected to two-pin photo-detectors, in a balanced photo-detection (BPD) unit.The OCT system is based on a low coherence interferometer with dynamic focus, where the coherence gate and focus gate are synchronized . This prThe OCT system is equipped with a 2-D GS which is comprised of two 1-D GSs with orthogonal axes; they provide a raster scan of the laser beam on the surface of the sample.Using the translation stage (TS), the optical path in the reference arm and the one in the sample arm of the interferometer are scanned together. An A-scan (in-depth reflectivity profile) is obtained for each of the positions of the laser beam on the frontal surface of the tooth. B-scans  are generated as raster scans by collecting many such A-scans from adjacent transverse positions; they provide therefore 2-D sections of the dental material in a plane sharing a lateral coordinate and the axial (in-depth) coordinate. B-scans are produced by the fast GS, driven in this setup with a triangular signal with a frequency of 500 Hz. Driving the GS with a triangular signal allows production of OCT images with less fly-back distortions than when using a saw-tooth signal , 26. OCT″  × 2″ positive system. The numerical aperture of this microscope objective determines the lateral size of the B-scans, which can be in this case adjusted continuously up to 10 mm.The second, slow GS positions the line of pixels generated by the fast scanner on the surface of the sample with a ramp signal with a frequency of 2 Hz. This can be repeated at different values of the second lateral coordinate, using bias voltages applied to the two GSs . The micAs discussed in the next section, for this dental application, B-scans of 2 to 3 mm in lateral size were enough to be considered in order to image the area of interest in the teeth. The wider B-scans along the lateral direction allow initial localization of the area of interest at the start of the procedure. By assembling successive B-scans, three-dimensional (3-D) reconstructions/volumes of dental constructions and dentures are obtained, as described in previous works . The basThe working protocol for this study has been approved by the Ethics Committee of the Victor Babes University of Medicine and Pharmacy of Timisoara. All experiments were conducted according to Romanian and European Union regulations.During the cavity preparation of each sample, an OCT 2-D slices/B-scans were generated. They revealed in real time the distances from the bottom of the drilled cavities to the pulp chamber. The 2-D slices were then combined into 3-D reconstructions, as shown in A validation of the OCT imaging procedure is shown in Figures On the 3-D OCT investigations, the RDT between the pulp chamber and drilled cavities was assessed, as demonstrated in However, the depth of a restoration preparation is often based on the extent of the decay and cannot in such cases be controlled by the dentist. Therefore, sometimes, during the drilling procedure, fractures can occur; they can penetrate the remaining layer between the cavity and the pulp chamber. Such occurrences have been documented in real time: in This process is shown further in This investigation clearly shows that by evaluating the current width of the remaining dental layer , the dentist can estimate the risk of fracture in real time during the drilling process. Based on such findings, the dentist can decide whether to use further endodontic therapy instead of a conventional filling procedure.After completion of the dental procedures (combined with OCT imaging), the accuracy of the RDT measurement through deep dentin cavity with OCT was verified for each sample. Thus, each tooth was finally sectioned in two halves and the RDT was measured directly. The values obtained confirm the results estimated during the OCT imaging. The final RDT value obtained using OCT for each sample was thus validated. The validity of the OCT monitoring, its capability to determine the internal topography of the tooth without distortions, was thus demonstrated.The knowledge of the internal dental anatomy is fundamental for the success of any dental therapy. In spite of the fact that every group of teeth presents similar morphological features, the individual variations in pulp chamber anatomy are a challenge for every clinician, with direct impact on the outcome of the dental therapy.Besides the issue of the different locations and shapes of the pulp chamber at different teeth and individuals, there are several reports on the existence of supplementary pulp horns. Their identification based only on retro-alveolar or panoramic X-rays is however difficult because the real 3-D structure of the pulp chamber volume cannot be inferred using a 2-D projection.Cone Beam Computer Tomography (CBCT) relies on radiations and so it is an invasive technique, therefore not suitable for dental routine treatment. CBSCT cannot distinguish microanatomical details smaller than 0.76 mm. As consequence CBCT is considered a complementary modality for specific applications rather than a replacement for 2-D imaging.Dental operating microscopy is efficient in all the steps of the endodontic therapy but provides magnification and illumination only when a communication between the dental cavity and the pulp chamber has been already established. This is a severe limitation for the common dental therapy and it would not fulfill the aim of this study.Orascope is a medical endoscope modified for treatment in the oral cavity. It helps to find cracks on the floor of the pulp chamber and extra canals more easily than with loupe spectacles. Compared with the dental microscope, its flexibility facilitates the application in the lateral tooth region. Additional intraoperative radiological checks can be avoided and the radiation load for the patients is thus reduced. However, many microendoscopic medical applications require small working diameters , which permit only about 3000 pixels for the image guide because of space requirements, while higher resolutions, of 10,000 to 50,000 pixels, would be needed. Moreover this technology offers only surface information, with no details related to the dentin floor depth, and therefore no internal cracks or fractures can be discovered. Above these limitations, the accidental communication between the cavity and the pulp chamber remains undiscovered.By contrast to such methods, OCT represents an alternative noninvasive technique to these shortcomings. OCT exhibits resolutions better than a few micrometers. The penetration depth in teeth is only 1 mm, because the hard tissue of the teeth decreases the backscattered signal strength. However, as demonstrated, even such reduced penetration depth is sufficient for guidance of dental procedures related to the drilled cavity and to the pulp chamber.Such methods are reviewed below. ex vivo study presented to an in vivo study, to assist dental procedures performed in the oral cavity [The results we have obtained proved that OCT can perform quantitative data retrieval of the relation between the drilled cavity floor and the pulp horns. Fast OCT real time acquisitions of over 1 Gigavoxel of data in a single second  are now l cavity . OCT unil cavity  to dental cavity . The nex ex vivo study presented illustrates the utility of OCT imaging employed in the training process of dentists performing dental procedures.In conclusion, the study demonstrated that OCT technology can be harnessed to assist cavity preparation that can lead to safer and more conservative results of dental procedures in real time. The in vivo investigations. The real time OCT monitoring proves promising in the prevention of accidental pulp exposure during deep caries excavation.It is also expected that such procedures can be extended to clinical environments for


Paramecium, the TZ is well structured with three distinct plates defined as the terminal, the intermediate and the axosomal plates. In this model, structural and molecular changes of the TZ are observed as anchored basal bodies become ciliated. Therefore, Paramecium appears to be a pertinent model to study the TZ at an ultrastructural level in correlation with its functionality.The Transition Zone (TZ) is defined as the most proximal region of the cilium overlapping with the most distal region of the basal body. This zone has been shown to play a crucial role in cilia biology since it is considered as the site of sorting of proteins that transit to cilia. Protein complexes housed at this zone are found mutated in MKS/NPHP ciliopathies. Although its organization varies from organism to organism, the TZ molecular composition and function are highly conserved. In Paramecium basal body cortex units. These units fit the cryo-electron tomography requirements allowing us their visualisation in native conditions at nanometric resolution.To reach this goal, we have developed a technique to isolate Paramecium units allow the observation of well-preserved basal bodies revealing the TZ with its three recognisable plates and the Y-links as well as at the proximal part of the basal body, the cartwheel and the radial spokes.First cryo-tomograms obtained on these Thus, studies of the consequences of TZ protein depletion at high resolution are now achievable by combining new isolation protocols and cryo-electron tomography.


Intrinsic motivations drive the acquisition of knowledge and skills on the basis of novel or surprising stimuli or the pleasure to learn new skills. In so doing, they are different from extrinsic motivations that are mainly linked to drives that promote survival and reproduction. Intrinsic motivations have been implicitly exploited in several psychological experiments but, due to the lack of proper paradigms, they are rarely a direct subject of investigation. This article investigates how different intrinsic motivation mechanisms can support the learning of visual skills, such as “foveate a particular object in space”, using a gaze contingency paradigm. In the experiment participants could freely foveate objects shown in a computer screen. Foveating each of two “button” pictures caused different effects: one caused the appearance of a simple image (blue rectangle) in unexpected positions, while the other evoked the appearance of an always-novel picture . The experiment studied how two possible intrinsic motivation mechanisms might guide learning to foveate one or the other button picture. One mechanism is based on the sudden, surprising appearance of a familiar image at unpredicted locations, and a second one is based on the content novelty of the images. The results show the comparative effectiveness of the mechanism based on image novelty, whereas they do not support the operation of the mechanism based on the surprising location of the image appearance. Interestingly, these results were also obtained with participants that, according to a post experiment questionnaire, had not understood the functions of the different buttons suggesting that novelty-based intrinsic motivation mechanisms might operate even at an unconscious level. Intrinsic motivations are motivations that drive human activities “for their own sake” [extrinsic motivations [wn sake” , for exaivations –5. From ivations –8.mechanisms. The first involves competence-based intrinsic motivations, linked to improving the agent's ability (competence) to modify the world through action [goals/tasks. Computational models have captured these mechanisms with algorithms that estimate the probability of successfully accomplishing goals. High motivation is generated when the probability is low, or, alternatively, while probability of success increases [stimuli [knowledge-based intrinsic motivations to distinguish them from competence-based intrinsic motivations [Prediction-based intrinsic motivations are those related to predictions of a possible future state [prediction-error and those associated with prediction error-improvement. For the former, motivation is generated by the magnitude of the error of the environment-model predictions [ovelty-based intrinsic motivations are elicited by objects, or object combinations, that have not been previously experienced and hence are not in the agent's memory. Computational models capturing these processes are typically based on the detection of anomalous/unfamiliar stimuli [Interdisciplinary research involving psychology, neuroscience, and computational modeling have highlighted at least three main classes of intrinsic motivation h action –13. In pncreases ,13. The [stimuli , and havivations . Recentlivations ,18. Predre state ,20. The re state ,21. To fdictions ; while f stimuli .mechanisms can play different functions/roles in cognition, but can also operate synergistically. For example, knowledge-based intrinsic motivation can lead the agent to form and select goals , while competence-based intrinsic motivation could guide the agent to learn the skills necessary to accomplish such goals [Note that these three types of intrinsic motivation ch goals .Neuroscientific research is starting to uncover the neural bases of intrinsic motivation mechanisms. Relevant for prediction-based intrinsic motivations and their role in learning motor skills, Redgrave and Gurney  argued tIntrinsic motivations have been implicitly exploited in several psychological experiments, including experiments on free visual choice with adults –37 and wFree-viewing ,44 and gThe current study directly investigated the effect of novelty-based and surprise-based intrinsic motivation on free exploration. Specifically, the experiment involved three conditions. The first condition investigated the effects of surprise on motivating saccades. Different levels of surprise were manipulated by letting a specific gaze direction pointing to either one of two button-like pictures to cause the appearance of a simple stimulus on a computer screen respectively at unexpected random positions or at a fixed position. The expectation was that the participants would experience a stronger motivating effect when stimuli appear at a random rather than at a fixed position as there would be a greater prediction error. The second condition investigated the motivating effects of content novelty of stimuli on foveation. Different levels of novelty were manipulated by letting a specific gaze direction pointing to either one of two button-like pictures to cause the appearance of pictures which were either always new on each trial, or consisted in simple unchanging stimulus. In this condition we expected a stronger motivational effect of novel stimuli. To evaluate their relative strength a third condition was used to directly contrast the comparative motivating effects of surprise and novelty.A total of 24 participants  took part in the current study. They gave their written informed consent to participate to the experimental procedure and to the subsequent treatment of data. Moreover, the study was approved by the ethic committees of the “Istituto di Scienze e Techologie della Cognizione, Consiglio Nazionale delle Ricerche”. The Open Gaze Tracker and Analyzer (OGTA) system developed by the authors was used to provide quantitative measurements of the participants' gaze. OGTA is a remote low cost eye tracking system based on off-the-shelf components and open source modular software designed for use in psychological experiments  without a significant reduction in accuracy. Blink or occlusion recovery was faster than 10 msec. During the calibration process, attractive circles were presented on the screen in a nine-point calibration sequence. The  coordinates (in pixels) of the nine calibration positions on the screen having a 1280x1024 resolution were: , , , , , , ,  and . The calibration procedure established an accuracy of 1 degree or less of visual angle, which corresponded to about a 2x2 cm area on the screen at a viewing distance of about 60 cm.http://www.nationalgeographic.com/) and from the Amsterdam Library of Object Images . One hundred animal and object pictures were used . The horizontal width of pictures was 400 pixels and the radius of the two button discs was 150 pixels. Areas of interest for eye tracking analysis were defined to match the position and size of the red discs and of the displayed images. Data analysis was performed with MATLAB. For each condition we used the Fourth-Spread statistical method to find and remove the outliers in the data-set. This method first identifies the boundaries of each of the quartiles in the data set. Then, it measures the fourth-spread (fs) as the distance between the lower and upper quartiles. The upper and lower outlier boundaries were set to 1.5 times fs above and below the median , or a blue rectangle (the “simple stimulus”). In addition, two red discs were presented, which served as “buttons” that could be “pressed” by the participants directing gaze to them. Animal and object pictures were taken respectively from the National Geographic Web Site  or a blue rectangle, at a fixed or random location on the screen, depending on the precise experimental configuration. A visual stimulus occurred immediately after a red disc was foveated, and stayed on the screen for 2.0 sec, during which the red buttons were disabled . After the image was removed, a new trial started where the participant could again trigger one of the two buttons to elicit the appearance of a new visual stimulus, until in any condition there had been 50 such trials, i.e. the buttons had been triggered 50 times.Latin square [The experiment was divided into three conditions corresponding to three different consequences of gazing at the left and right buttons: 1) the first condition tested surprise-based intrinsic motivation (SUR condition); 2) the second condition tested novelty-based intrinsic motivation (NOV condition) and (3) the third condition compared the two motivations (SUR-NOV condition). The order of the conditions was balanced across participants. In the same way, the effects of the two buttons (left or right) were also balanced across participants: we applied a n square ,53 desig the firs the secoLooking at one of the buttons caused a simple picture  to suddenly appear at a position equidistant between the two red buttons: this position was always the same in different trials. Looking at the other button caused the same blue rectangle to appear but at different random locations on the screen in different trials. Our prediction for this condition was that the ‘random location’ button would be more attractive for the participants because the action effect would be unpredictable and therefore violate their expectations. This condition should act mainly through the prediction-based intrinsic motivation mechanism. The putative brain mechanisms responsible would likely involve the retinotopic representation of luminance changes in the superior colliculus triggering phasic dopamine responses that, because the stimuli were presented at random locations, could not be predicted.In this condition the direction of gaze to the two buttons caused the appearance of pictures always in the same place between the two red buttons. However, the fixation of one of the two buttons always caused the appearance of the same blue rectangle, while the other button caused animal/object pictures that changed from trial to trial. In this condition our prediction was that the button causing the appearance of different pictures would be more attractive as it would provide novel content information. The brain mechanisms that might underlie this process would be the capacity of hippocampus to detect complex novel patterns.In this condition one of the buttons triggered the appearance of the blue rectangle at random locations, while looking at the other button caused the appearance of a changing animal/object image at a fixed location. This condition aimed to reveal which of the two surprise/novelty motivation mechanisms is stronger.The two types of stimuli used in the experiment stress the operation of one intrinsic motivation mechanism but do not completely eliminate the operation of the other. In general, the experimental procedure was designed, (a) to balance the effect of one of the two mechanisms for the two buttons when not relevant (condition 1 and 2); (b) to test the relative strength of one system versus the other (condition 3).Thus, in condition 1, which probed prediction-based intrinsic motivation, the presence of a novelty detection system would be balanced across the two buttons as same blue rectangle was elicited by both. Similarly, in condition 2, which probed the novelty-based intrinsic motivation, the location at which the novel and familiar images were presented remained constant. Finally, in condition 3, which contrasted the surprise- vs. novelty-based detection systems, while the two buttons may engage both systems, each would produce effects that would activate one of the systems more strongly. For example, novelty detection would be triggered by the first appearance of the blue rectangle but would rapidly habituate given the comparative simplicity of the picture. However, the surprise detection mechanism would be expected to remain highly engaged by pictures continually appearing in random locations. Alternatively, when an animal/object picture first appears it should engage the surprise detection system. But again, as the new pictures always appear at the same location this element of surprise should habituate rapidly. The novelty detection systems should, however, be continually stimulated as successive pictures were always novel.across images was zero, and so each pixel value was perfectly predictable after the first experience. Moreover, the entropy of pixels within each image is also zero, having them always the same value and so each pixel perfectly predicts all others. This enables the participants to remember perfectly the blue rectangle after the first experience. This means that the novelty of this stimulus  and with the independence between pixels . From tception, ) is miniAt the end of the experiment the relative frequencies of gaze shifts to the two buttons was evaluated using the t-test and a one-way analysis of variance (ANOVA). In particular, for each condition , we performed a t-test to rule out the null hypothesis for which data representing the frequency of gazing at a button were a random sample from a normal distribution with mean 25  against the alternative possibility that the mean was not 25. The frequencies of gaze at the two buttons were fully dependent because the number of total trials was fixed (50), so it was necessary to perform the t-test on the data related to only one button to avoid an overestimation of the statistical significance of the results. The one-way ANOVA was performed to check if there was a significant difference in the gazing behavior of participants that comprehended the experiment versus those that did not comprehend it. In both type of tests a result was considered significant if the p value was less than 0.05. The comprehender/non-comprehender groups were determined on the basis of the post experiment questionnaire. A participant was classified a “comprehender” if he/she understood that looking at one of the buttons triggered the appearance of a new picture  or a blue rectangle, in a fixed or a random position.In this condition looking at one button caused the appearance of a blue rectangle in a random position in the screen, while looking at the other button caused the appearance of a blue rectangle always at a fixed position between the two buttons. Contrary to our expectation, data illustrated in prediction error. Indeed, the results show how the blue rectangle appearing in the fixed rather than the random position tends to motivate gaze to be directed at its related button. A possible explanation of this result is that the fixed location of the stimulus allowed participants to learn to predict where it would appear. This suggests that participants may be motivated more by the improvement of the prediction error rather than by the prediction error [inner mechanism that could drive the behaviour of infants to seek proper levels of experience novelty, thus avoiding overly simple or complex events [This result contradicts our prediction on the presence of a surprising feature of the stimulus based on its unpredictable presentation location sufficient to activate an intrinsic motivation mechanism based on on error . This poon error . Similaron error . While tx events . Infantsx events .Future work could further investigate the prediction error improvement mechanism possibly involved in our experiment by manipulating the predictability of the items' appearance. For example, it could contrast a condition where a stimulus appears in a fixed position with a condition where it appears in a sequence of positions having a regular pattern .In this condition, the contrast caused by gazing at the two buttons was between the appearance of same blue rectangle and different pictures , both always appearing in the same fixed position. Our expectation was that the pressure of the button causing the appearance of novel pictures would have been more motivated. The data shown in The results in In this condition the comparison was between the appearances of the simple picture (blue rectangle) in different locations with different complex pictures appearing in a fixed position. This condition aimed to contrast the intrinsic motivation mechanisms based on surprise and novelty. Given the result involving surprise was negative, the current condition confirmed the stronger influence of the intrinsic motivation based on novelty. The results shown in As far a we know, this is the first empirical study to directly investigate the interplay between two intrinsic motivation mechanisms, one based on a familiar stimuli appearing at surprising locations and the other based on novel stimuli appearing at a familiar location. The contrast between these two conditions was in their ability to intrinsically motivate the acquisition and maintenance of gaze behavior directed to two visual targets. The demonstrated predominance of novelty-based intrinsic motivation suggests a possible involvement of the hippocampus . The comThe work also highlights the importance of entertaining the possibility that intrinsic motivation mechanisms involving stimulus features including novelty/familiarity/surprise might operate at an unconscious level. This is a second issue that deserves further investigation.Finally, the experiment shows that the gaze contingency paradigm can be used effectively to investigate the influence of intrinsic motivations on the acquisition of visual skills. Moreover, the current study provides a sound base from which the same processes can be investigated in babies, with a view to identifying a developmental profile of how intrinsic motivations can drive learning and behavior in different age groups.


Agent-based models (ABMs) have been widely used to study socioecological systems. They are useful for studying such systems because of their ability to incorporate micro-level behaviors among interacting agents, and to understand emergent phenomena due to these interactions. However, ABMs are inherently stochastic and require proper handling of uncertainty. We propose a simulation framework based on quantitative uncertainty and sensitivity analyses to build parsimonious ABMs that serve two purposes: exploration of the outcome space to simulate low-probability but high-consequence events that may have significant policy implications, and explanation of model behavior to describe the system with higher accuracy. The proposed framework is applied to the problem of modeling farmland conservation resulting in land use change. We employ output variance decomposition based on quasi-random sampling of the input space and perform three computational experiments. First, we perform uncertainty analysis to improve model legitimacy, where the distribution of results informs us about the expected value that can be validated against independent data, and provides information on the variance around this mean as well as the extreme results. In our last two computational experiments, we employ sensitivity analysis to produce two simpler versions of the ABM. First, input space is reduced only to inputs that produced the variance of the initial ABM, resulting in a model with output distribution similar to the initial model. Second, we refine the value of the most influential input, producing a model that maintains the mean of the output of initial ABM but with less spread. These simplifications can be used to 1) efficiently explore model outcomes, including outliers that may be important considerations in the design of robust policies, and 2) conduct explanatory analysis that exposes the smallest number of inputs influencing the steady state of the modeled system. Socioecological systems are perpetually dynamic and nonlinear We argue that systematic evaluation of ABM uncertainty should comprise a joint quantification of model output variability and its sensitivity to inputs  - Recognized as important for scientific understanding, quantitative UA-SA has been employed in a number of non-ABM studies on socioecological systems. Examples include ecological modeling of ecosystem vulnerability to climate change exploration of extreme system behavior. Although highly unlikely, boundary cases may result in radical changes of significant consequence to society and/or the environment. On the other hand, scientific explanation requires considerable accuracy, which can be achieved through reducing output variability in order to improve model performance.Quantitative UA-SA in ABMs can serve many purposes exploratory model that allows scientists to simulate low-probability but high-consequence events that may be of high policy relevance. Second, we build a more explanatory model that provides the means of describing the system with higher accuracy. Specifically, we postulate that the explanatory power of a model lies in illuminating the core underlying processes In the next sections, we describe how quantitative UA-SA can be used to build ABMs for policy analysis and exploration. While still nascent, a comprehensive UA-SA have been applied to ABMs in a few previous studies UA produces a distribution of model results . It requCommonly, SA involves modifying the value of one factor (while keeping the other factors constant) and observing the effects of this change on model results. This method, referred to as one-parameter-at-a-time (OAT) i is the variance of Y due to the variability of factor i alone, and VCi is the conditional variance due to all model factors except i. The sum of all S indices (ΣS) is the fraction of output variance that can be explained by the individual factors alone. Therefore, the formula:I) between the factors. This succinct measure of interactions can be further analyzed using the ST indices, which provide information about the total (first and higher order) influence of each factor on output variance. For more details on variance decomposition, the reader is referred to Saltelli et al. Our global SA uses model output variance decomposition in which the variability of the area of fallow land (resulting from farmer agent decision making) is decomposed (partitioned) and distributed among model factors evaluated in various combinations explanatory power conception of modeling (exploratory power (embodied in variance of a given output) equal to this model's baseline implementation  pairs are quantified as ratios of the conditional output variances to the total variance and, thus, measure the relative contribution of each factor to output variance . Factorsmodeling . The majentation .We use quantitative UA-SA for land use model simplification and factor prioritization. The goal is to build a simpler representation of an ABM with two distinct objectives: policy analysis that would benefit from exploratory modeling Published research suggests that farmers' decision to participate in a land conservation program is driven by both financial and nonmonetary drivers land tenure, operator's retirement, and the value of production on a farm; described in later sections). The FA is then assigned to a parcel.In the ABM reported herein, CRP enrollment is simulated based on well-defined federal regulations decision rules) called Ordered Weighted Averaging - OWA density of enrollment in its neighborhood, measured as the ratio of CRP-enrolled neighbors to the total number of neighbors within 0.5 km from the parcel.As a first step, the FA calculates their willingness to enroll in the CRP based on decision criteria  including financial motives and nonmonetary drivers. To calculate the willingness to enroll, we apply a group of aggregation operations  of them based on the environmental benefit index (described in the following section), the signup budget, and the DAP. Next, FSA announces the signup results leading to land use change from agriculture to fallow. In sum, the location and area of fallow land results both from the FAs' decisions to participate and the FSA's decision to accept their offers. The process of CRP signup is repeated annually for ten years (minimum CRP contract length). Land use change maps constitute the output of the model. They are summarized into the total area of fallow land. This scalar is used in the UA and SA.If an FA's willingness to enroll exceeds an empirically derived threshold, the agent selects a The ABM uses a number of factors, some that are readily available and some that we derived from auxiliary resources, including land uses obtained from 2010 cropland raster layers Ranking of CRP offers is based mainly on their Environmental Benefits Index (EBI) values. EBI is a composite index based on multiple rated criteria describing benefits for wildlife, water quality, soil erosion, long term maintenance of installed vegetation, and air quality EBI can be quantified in many different ways, resulting in substantial uncertainty. Consequently, we used alternative conceptions of the benefits , weighedStatistical and econometric studies of CRP enrollment point to five major categories of independent variables used to predict participation in land conservation programs: farm, household, and environmental characteristics; government assistance; and farmers' attitude and perception a priori set of candidate multiple regression models to understand farmer participation in the CRP based on results of prior studies about farmer participation in land conservation programs. Using a multimodel inference analytical approach based on the Akaike information criterion (AIC), models with relatively low AIC values were considered the most parsimonious, balancing bias and variance of model predictions We developed an Given the CRP enrollment procedure and the available data, we identified nine factors for the ABM. Seven factors are attributed to the FA and the remaining two to FSA . The thrEmpirical data for other factors were only partially available. Consequently, we used a uniform PDF for the other model factors http://www.python.org/) and the  indices were computed using the SimLab software package for uncertainty and sensitivity analysis (http://ipsc.jrc.ec.europa.eu/?id=756). Statistical regressions were completed utilizing R, version 3.0.2.Our simulations include three computational experiments. In experiment one , our base scenario , we run fallow land, when compared to our input land use maps. Example results from EXP1 are depicted in The results of our ABM simulations are land use maps with one additional category, total fallow land area - AREA). To explore the variability of CRP enrollment, we performed UA by examining the distribution of the total area enrolled in CRP  produces a distribution much more centered around the mean when compared to the baseline. Consequently, the simplified and refined ABM used in EXP3 can be used in explanatory analysis of the social, economic, and ecological processes associated with CRP participation. The following section explains the details on how we arrived at these two ABM simplifications.We used the variance to evaluate the degree of AREA variability. As expected, the variances of EXP1 and EXP2 are approximately equal. Consequently, the simplified model used in EXP2 can be used in UA alone does not provide any information about the influence of individual factors on AREA variability. Without SA it would be impossible to build the simpler yet equivalent versions of our ABM. By performing the decomposition of AREA variance, we can identify factors in the initial version of the ABM that can be either reduced without the loss of ABM exploratory power (EXP2), or refined if our objective is to explain the processes (EXP3).exploratory model , to demonstrate how the behavior of our ABM changes when, instead of fixing the negligible factors, we do so for the most influential factor. This scenario imitates a situation in which we obtain more accurate data on the most sensitive factor of the model. There was a significant reduction in AREA , and altOur combined quantitative UA-SA framework serves as a tool for better-informed ABM building. It leads to equivalent but simpler representations of a given socioecological system. Output uncertainty can be greatly reduced if more effort is put into improving the quality of data on the most influential factors (factor prioritization) through additional field studies, surveys, or auxiliary databases. However, the UA-SA framework also has limitations due to two design aspects: factor distributions and the type of output variables used . A different output variable  might point to a different set of influential factors. For example, the use of spatial metrics applied to output land use change maps The ABM presented here is of limited use for natural resource management practice. Data for most of the factors are either simulated or come from secondary sources and some of the mechanisms are poorly defined. Future model improvements will require surveys of and interviews with farmers and government officials. The recent decline in CRP enrollment suggests that increasing crop prices and government subsidies may play a significant role in the extent of land conservation ABMs have distinct advantages over other modeling approaches due to their abilities to couple human and natural systems, to incorporate micro-level behaviors among interacting agents, and to understand emergent phenomena due to these interactions. Their use thus far has been primarily by researchers for descriptive and predictive purposes We also used the quantitative UA-SA approach to improve the ABM's policy relevance. Lempert Interest in the study of complex socioecological systems or coupled human and natural systems has risen concomitantly with the recognition of profound challenges in the Anthropocene including climate change, biodiversity loss, land use change, alteration of nitrogen and phosphorus cycles, and the depletion of freshwater Code S1Pseudo code of the main routine in the Agent-Based Model of Participation in the Conservation Reserve Program. CRP: Conservation Reserve Program, FALLOW: total area converted to fallow (in pixels), FSA: Farm Service Agency, OWA: ordered weighted averaging decision rule, SITE: a pixel belonging to a given farm parcel. Factors are presented using uppercase bold fonts.(DOCX)Click here for additional data file.


Several studies have presented technological ensembles of active knee systems for transfemoral prosthesis. Other studies have examined the amputees' gait performance while wearing a specific active prosthesis. This paper combined both insights, that is, a technical examination of the components used, with an evaluation of how these improved the gait of respective users. This study aims to offer a quantitative understanding of the potential enhancement derived from strategic integration of core elements in developing an effective device. The study systematically discussed the current technology in active transfemoral prosthesis with respect to its functional walking performance amongst above-knee amputee users, to evaluate the system's efficacy in producing close-to-normal user performance. The performances of its actuator, sensory system, and control technique that are incorporated in each reported system were evaluated separately and numerical comparisons were conducted based on the percentage of amputees' gait deviation from normal gait profile points. The results identified particular components that contributed closest to normal gait parameters. However, the conclusion is limitedly extendable due to the small number of studies. Thus, more clinical validation of the active prosthetic knee technology is needed to better understand the extent of contribution of each component to the most functional development. A prosthetic knee is the key component of a transfemoral prosthesis prescribed to above knee amputees as it replaces the functional joint. The development of prosthetic knee systems has increasingly enabled amputees to perform activities of daily living (ADL). The variation in requirements of daily behavior of the persons who lost their lower limb has encouraged scientists to continuously improve the prosthetic knee mechanisms. To understand the recent advancement of the knee technology, different knee types are classified according to its functional mechanism. The technical capability and performance of a knee prosthesis are instrumental in facilitating a walk that more closely resembles that of an able-bodied individual's biomechanics and thus increases amputee walking energy efficiency. Therefore, the extent to which the amputee can achieve normal gait biomechanics highly depends on the appropriate use and integration of sensory systems, actuators, and control scheme.Passive knee devices have fixed impedance that is provided from either pneumatic or hydraulic systems, in which the devices' friction might change according to the walking speed of the amputees . Unlike Another type of passive knee device is the single axis knee, which utilizes a simple pivot mechanism. It is robust, lightweight, and relatively cheaper than other knee systems that are commonly in use. However, amputees have to use their own muscles to maintain the device stable at standing, as it is not equipped with a stance control function. Thus, users often use manual lock to compensate for lack of stance control and utilize the available friction to prevent the leg from overspeeding during the forward swing when moving into the next step . PolycenAnother type of knee prosthesis is the powered prosthetic knees that have the capability to assist amputees to perform level walking, ramp descent, stair descent, and standing and detect instances of stumble . HoweverTo improve the mobility of the knee prosthesis system, they incorporated the ability to restore user stability. The knee prosthesis was referred to as active device, as it is capable of delivering active response to prevent the amputee from falling due to obstacles. One example of this active powered knee can be seen in the stumble detection feature in which real-time stumble can be detected using three separate accelerometers on the prosthesis. Ten subjects were employed to validate the stumble detection through these signals and all 19 stumble responses were correctly identified . FurtherAdaptive dissipation knees refer to devices that can regulate the impedance by adjusting the hydraulic valves such as the Otto Bock C-Leg or the devices that use magnetorheological fluid such as Rheo knee. Microcontroller on board can regulate the impedance by tuning hydraulic valve, that is, the orifice that rectifies the flow rate. Adaptive knees adjust the resistance to control the knee after calculating the comparisons between steps and monitoring the position, velocity, forces, and moments of the prosthetic knee . To diffIn general, the working principle of a smart or active device involves the integration of intrinsic computational sensors, whereby the sensors detect the physical performance of the system and this corresponds to real-time alterations in the actuator. However, an embedded controller adjusts and coordinates the knee movement accordingly . The firOne study compared the kinetics and kinematics performance of the C-Leg and the Mauch SNS knee prosthesis during gait . The stuDespite all the commercially available and research-based transfemoral prosthesis inventions, there has not been an overall systematic analysis that directly relates the technology employed with its users' functional walking outcomes. This study was designed to establish an understanding of the existing scientific studies that addresses the development and outcome of electronic prosthetic knee for transfemoral amputees based on a biomechatronics approach . In partPublished articles were searched using different databases, namely, the Academic Source Premier at EBSCOhost (© 2012 EBSCO Industries), ACM (Association for Computing Machinery) , Biological Abstracts at Ovid , Cambridge Journals Online (Cambridge University Press 2012), Emerald , IEEE Xplore (© Copyright 2012 IEEE), ScienceDirect (Copyright © 2012 Elsevier B.V), SpringerLink , PubMed (Copyright 2012 NCBI), and Taylor & Francis Journals (© 2012 Informa Plc). Studies were limited to English language or other languages that were translated to English. The criteria of focus were as follows: (1) sensors, (2) actuators and transmission systems, and (3) control techniques. Keywords that were used to identify suitable articles were (a) lower prosthesis, (b) prosthetic leg, (c) prosthetic knee, (d) knee prostheses, and (e) active knee. To recognize appropriate articles and ensure that all the chosen articles satisfied the inclusion criteria as defined by the key search terms, the authors screened the title and abstracts of all computer-generated search results articles. θ), moment or torque (T), and power (P), were identified [Only studies that reported clinical evaluation were included as they described the performance of the knee parameters during gait cycle. The performance parameters were later used in the efficacy assessment of the prosthetic knee devices by comparing them with the normal parameters. Other studies were excluded, as illustrated in entified  to repreθmax. stance), minimum stance angle (θmin. stance), maximum swing angle (θmax. swing), and minimum swing angle (θmin. swing). These points that were chosen as the range of angle sensors were identified and selected based on the four angle parameters. To predict the required torque of the knee actuator, the following parameters were identified: maximum stance flexion (Tmax. stance flex.), minimum stance extension (Tmin. stance ext.), and summation of positive and negative torque during swing phase in terms of the area under the curve (TArea). To determine the functional performance of the prosthesis in terms of power, the full gait cycle power's profile was referred to the maximum power during propulsion (Pmax. propulsion) and maximum power during swing (Pmax. swing) selected as the comparable parameters. The power values provided an indication of the amount of generated and absorbed mechanical power during propulsion and swing phases, respectively [Four parameters were extracted from the knee angle profile: maximum stance angle ::1)θ=|θsθ=|θs(1)θQ, is shown inQj is the value of each parameter Q in a particular study j, Q refers to each of the nine subparameters , N is the number of the subjects participated in each study j, and Z is the number of studies.A general weighted average equation to calculate the relative deviation of each knee parameter, θmax. stance for one of the studies employing angle sensor (S1p) is illustrated as in  18°ar study  and 20° shown in  and the As the current methodology will be useful in the development of active prosthetic knee devices, attention was given to the intrinsic thus controllable knee kinematic and kinetic parameters from different knee components such as sensors, actuators, and control algorithm. After exclusions, 53 studies were identified as being relevant to this research on microcontroller-based transfemoral prosthesis. Out of those, 24 studies reported the utilization of interactive extrinsic techniques and these were excluded. Those extrinsic techniques entailed that the system offered a level of communication between the user and the device, which may encompass afferent and efferent communication. This may include EMG signals from the muscles or other efferent systems that allow communication from the brain to be integrated in the control loop . ControlQ values closest to 1 indicate the least deviation from normal; thus components with values closest to one are perceived to be the most successful components in producing a close to normal gait.Q values closest to 1 for different knee subparameters were highlighted, and asterisk values. Therefore, values closer to normal for every subparameter indicate the effect of technology in terms of sensors, actuators, and control techniques for future development of prosthetic knees. The nine subparameters of the kinematics and kinetics for the knee during the gait cycle were presented with respect to the technology that was utilized in each prosthetic knee device. The use of angle sensor potentiometer helped to increase the behavior accuracy of most of the knee parameters, as shown in the first row of The development of lower prosthesis that mimics natural knee movement is a challenge, especially when selecting an actuating mechanism that is capable of delivering stability and stiffness to the prosthesis.This study focused on the overall effect of each technology component, namely, sensors, actuators, and control technique that were incorporated in the various reported active prosthetic knee systems for transfemoral prosthesis. One might argue that it might be inaccurate or nonrepresentative to evaluate single components such as sensors, actuators, and controllers independently, as they all work as a whole system. In other words, the end gait performance of the system is a product of the whole synergy and it might not be accurate to claim that a better or worse performance is the singular effect of each component. Nevertheless, most components were reportedly used in multiple systems as presented in the studies retrieved. This allows one to deduce the potential performance of each component when it is integrated as part of any other system. On the other hand, many other microcontroller-based prosthetic knee systems have been developed that did not report any gait-related kinetics and kinematics performance, such as the Ossur Power Knee, Rheo Knee, and its combination with the Proprio Foot adaptive ankle system . This haThis paper included 7 studies based on five technologies. The five technologies out of the seven retrieved studies reported utilization of different actuation mechanisms, namely, (1) series elastic actuators , 37, 38,Agonist-antagonist active knee prosthesis , 38 contA linear step motor actuating four bar linkages was used to develop active knee prosthesis utilizing robotic control theories in the strategy of controlling the prosthesis. Using linear motor reduces the size and the weight of the system , while iThe Vanderbilt leg  was deveWhile it is important to identify and evaluate the performance of each sensory, actuation and control element of a powered knee system, a more crucial element in designing the powered lower prosthesis is to build the powered knee joint that has the capability of performing functional tasks such as sit-to-stand and stair climbing. Researches nowadays are investigating better prosthetic devices to assist the amputees during sit-to-stand and stair climbing. The functional capability that requires modulation of the power output of the limb should be achieved with the knee joint to contain the motorized actuators, sensors, and the controller. The following sections discuss the components of various technologies utilized in the retrieved studies.A prosthetic knee system requires many input variables to perform optimally; thus, all utilized sensors were discussed in the current study. To select appropriate sensory systems for the knee devices, the control variables have to be determined. Adaptive-control of prosthetic knee  was repoQ values at most of the torque and propulsion power. However, its kinematic performance was only acceptable for maximum stance and maximum swing angles. On the other hand, the technology that uses position sensor or hall sensor (S4) has shown reliable results for the knee angle parameters and only some torque and power measurements, that is, minimum knee stance torque and maximum knee propulsion power.Angle sensor potentiometer (S1p) has reported relatively acceptable values according to the nine subparameters of the knee . The potBoth angle sensor and moment or torque sensors play a prominent role in developing an active knee. In order to record heel strike and toe-off to provide information about walking phases, FSR is used, located directly below the prosthetic foot. Furthermore, it is low in price, relatively thin, small, and produces analog-based signal . The FSRThe natural knee produces an internal flexor moment, due to contraction of the hamstrings, which prevents hyperextension at the end of the swing phase. As the knee starts to flex, concentric contraction of the hamstrings, as well as the release of energy stored in the ligaments of the extended knee, results in short-lived power generation . For ampQ values (Q values.All actuator types are listed with different Q values . The motQ values, especially the swing phase knee angles, but only 1 study [Control strategy is considered as the most critical part in a power prosthetic knee; control strategy is not needed in nonpowered prostheses. Impedance control algorithm was the most commonly used control strategy, in which the torque generated is adapted to the produced knee angle. This mode of control ensures that the knee joint generates torque that is suitable for each gait phase , 41, 43. 1 study  reportedFor further enhancement of the prosthetic knee movement, all possible sensors that detect different knee variables should be in the prosthetic knee structure containment. The problem concerns not only the selection of a specific sensor, actuator, or control method but also the integration and synchronization between various components. By reviewing the knee power results for both normal and prosthetic knees, as shown in Figures Another conclusion that can be made in terms of the knee power as one of the important parameters which affect the knee performance is illustrated in One of the critical considerations in constructing lower limb prosthesis is the weight of the prosthesis. Nowadays, researchers try to optimize the weight during the development of the prosthetic knee. In addition, accompanying devices and weight reduction, strength and functionality are usually the primary goals in prosthetic fabrication. Metal is mainly used for rotating components whereas aluminum as an alternative to steel is used conservatively for smaller components. Titanium is more expensive; however, due to its biocompatibility, it is considered for some aspects of prosthetic devices .Lower limb prostheses need to support the body weight during the stance phase and must prevent the knee from sudden joint flexion . LighterAmputees can produce energy for their prosthetic leg by utilizing active energy harvesting device in the system. Such techniques may decrease the device's dependency on a battery and therefore may reduce the overall weight of the prosthesis. Incorporating new kinds of light material such as thermoplastics within structural components is a breakthrough in prosthesis fabrication . Rigid pProsthetic foot is considered one of the prosthetic knee components which may have affected the biomechanical outcomes at the knee joint . Thus, aThe sensors and actuators that are incorporated in the knee system need electrical power source for their operation and the power supply is another crucial component in microcontroller-based prosthetic knee devices. The power sources that are mentioned in the available studies are listed in x- and y-axes is a potential solution. Moreover, during stair climbing or slope walking activities, the sensors would be reliable in delivering instantaneous signals to the controller to adapt to the prosthetic knee movement. From a biomechatronics point of view, the integration of sensors and electric actuators such as electric motors is easier in terms of communication and interaction due to the same domain of electrical operating signals. This reduces the need for additional hydraulic or pneumatic sources.In order to achieve a more realistic prosthetic knee system for transfemoral amputees, additional sensors might be integrated in the knee system to recognize the intention of the amputee's movement. Combining gyroscopes with an accelerometer to recognize the orientation and acceleration of the knee joint in The control of lower limb prosthesis presents another significant challenge, especially during transition between phases. One example was illustrated in robotic ankle device that was capable of adapting its mechanical movement according to different walking conditions , 53. TheBy using robotic control algorithm, in which the controller supervises the trajectory of the joint position, it is possible to improve the ability of prosthesis to mirror the performance of a healthy leg profile . IteratiIn this paper, the efficacy of adaptive dissipative or powered-based prosthetic knee systems was reviewed based on the contribution of the sensor, actuators, and control technique towards the production of a normal gait profile. From the 7 retrieved studies, the knee's potentiometer was identified as the most commonly used sensor while its performance was comparably reliable as the FSR, moment sensor, and hall position sensors in measuring knee parameters. The DC electric motor connected to a ball screw mechanism was identified as the most used actuator with most reliable performance for the development of the powered or motorized prosthetic knee. These evaluations were performed by comparing the gait cycle behavior of the prosthetic knee systems with the normal gait cycle in terms of the nine subparameters of the knee. Minimum deviation from normal gait parameters was also derived from the application of the impedance control method, which in turn was suggested as the best technique for controlling prosthetic knee during different gait phases.Finally, the biomechatronics design approach was based on integrating the prominent sensors, actuators, and control techniques that were identified within this review; smart active prosthetic knee systems might be developed to strategically integrate all the identified components. Additional sensors could be embedded within the prosthetic knee system to enhance the interaction between the user and the environment. An alternative technique can be made by incorporating sensory system embedded inside the socket wall to receive direct information from the residual limb and further enhance the interaction between the user and the device as well as user intention. Furthermore, the knee controller could monitor the knee movement and intention based on contraction and retraction of the residual muscles, as well as the resultant force experienced at the stump from the body and the ground reaction forces. Similarly, the user can produce some of the electrical power required by using active elements such as piezoelectric material to produce the amount of electrical power needed to operate the electronic devices . UltimatBased on the current state of literature, it can be safely concluded that most research-based studies have not clinically validated performance with the real intended users. Therefore, identification of the best technology can only be limitedly made. This highlights the need for more clinical trials to be performed for the newly developed prosthetic knee systems to provide validated assessment of the prosthesis, especially in mimicking the biological gait across range of modes such as stair ascent and descent and ramp or uneven terrain walking.


Thus, the functional complex network inferred from activity patterns is a promising way to unravel the interplay between structure and functionality of neuronal systems. Here, we monitor the spontaneous self-sustained dynamics in neuronal cultures formed by interconnected aggregates of neurons (clusters). Dynamics is characterized by the fast activation of groups of clusters in sequences termed bursts. The analysis of the time delays between clusters' activations within the bursts allows the reconstruction of the directed functional connectivity of the network. We propose a method to statistically infer this connectivity and analyze the resulting properties of the associated complex networks. Surprisingly enough, in contrast to what has been reported for many biological networks, the clustered neuronal cultures present assortative mixing connectivity values, meaning that there is a preference for clusters to link to other clusters that share similar functional connectivity, as well as a rich-club core, which shapes a ‘connectivity backbone’ in the network. These results point out that the grouping of neurons and the assortative connectivity between clusters are intrinsic survival mechanisms of the culture.The analysis of the activity of neuronal cultures is considered to be a good proxy of the functional connectivity of  assortative mixing in networks' language. This particular choice of connectivity correlations must be rooted to basic survival mechanisms for the neurons constituting the culture.The architecture of neuronal cultures is the result of an intricate self-organization process that balances structural and dynamical demands. We observe that when the motility of neurons is allowed, these neurons organize into compact clusters. These neuronal assemblies have an intrinsic synchronous activity that makes the whole cluster firing at unison. Clusters connect to their neighbors to form a network with rich spontaneous dynamics. This dynamics ultimately shapes a directed functional network whose properties are investigated using network descriptors. We find that the networks are formed such that preference in connectivity between clusters is based on the similarity between their activity, a property that is called  The theory of complex networks One of the major challenges in biology and neuroscience is the ultimate understanding of the structure and function of neuronal systems, in particular the human brain, whose representation in terms of complex networks is especially appealing C. elegansNetwork theory and its mathematical framework have provided, through the analysis of the distribution of links, statistical measures that highlight key topological features of the network under study. These measures have facilitated the comprehension of processes as complex as brain development assortativity coefficient, which quantifies the preference of a node to attach to another one with similar (assortative mixing) or dissimilar (disassortative mixing) number of connections C. elegansA network measure that has recently caught substantial attention is the The assortativty coefficient is usually calculated through the Pearson correlation coefficient between the unweighted degrees of each link in the network in vitro preparations in the form of neuronal cultures have been introduced given their accessibility and easy manipulation clusters that connect to one another To better understand the importance of these network measures in describing neuronal networks, mesoscopic scales The formation of a clustered architecture from an initially isotropic configuration is an intriguing self-organization process In this work we use spontaneous activity measurements in clustered neuronal cultures to render the corresponding directed functional networks and study their topological properties. We introduce a novel theoretical framework that uses the propagation of activity between clusters as a measure of “causality”, although strictly speaking we should refer to as a sequence of delayed activations, giving rise to functional connections that are both directed and weighted. Based on this weighted nature of the network, we propose a new measure of assortativity that explicitly incorporates the weight of the links. We observed that all the studied functional networks derived from clustered cultures show a strong, positive assortative mixing that is maintained along different stages of development. On the contrary, homogeneous cultures tend to be weakly assortative, or neutral. Finally, in combination with experiments that measure the robustness of network activity to circuitry deterioration, we show that the strongly assortative, clustered networks are more resistant to damage compared to the weakly assortative, homogeneous ones. Our work provides a prominent example of the existence of assortativity in biological networks, and illustrates the utility of clustered neuronal cultures to investigate topological traits and the emergence of complex phenomena, such as self-organization and resilience, in living neuronal networks.clustered cultures that evolved quickly (day in vitro (DIV) We used rat cortical neurons in all the experiments. As described in  quickly . By day The example shown in We monitored spontaneous activity in the clustered network through fluorescence calcium imaging . Fluoreslity see . Activitbursts these fast sequences of clusters' activations. Young cultures  2 the culture encompasses dozens of small aggregates that coalesce and grow in size as the culture matures. Spontaneous activity and connections between clusters were observed by DIV Upon plating, the absence of adhesive proteins in the glass substrate rapidly favored cell-cell attachment and, gradually, the formation of islands of highly compact neuronal assemblies or changing medium  provided a layer of adhesive proteins for the neurons to quickly anchor upon seeding, leading to cultures with a homogeneous distribution of neurons over the substrate. The remaining steps in the preparation and maintenance of the cultures were identical as the clustered ones, i.e. we used the same nominal neuronal density for plating, we included PDMS pierced molds to confine neuronal growth in cavities in vitro (DIV) To measure the spontaneous activity in the clustered networks we used cultures at day 2, 2, Activity in neuronal cultures was monitored through fluorescence calcium imaging The observation chamber was mounted on Zeiss Axiovert inverted microscope equipped with a high-speed CMOS camera (Hamamatsu Orca Flash 2.8). We used an objective of The fluorescence signal of the clusters' spontaneous activity was recorded with the software Hokawo 2.5, provided by the camera vendor (Hamamatsu Photonics). We used acquisition speeds in the range et al.Measurements in homogeneous cultures were carried out in the same way, with the only difference that the recording speed was increased to We considered two groups of resilience experiments. In a first group, we monitored the gradual degradation of network activity due to photo-damage. In a second group, we measured the decay in activity as a consequence of the gradual weakening of the excitatory connectivity.In the first group of measurements, we first considered a clustered culture and measured its spontaneous activity uninterruptedly along 2 hours, with neurons continuously exposed to a light radiation networks  indicateIn the second group of measurements, we compared the change in activity between a clustered and a homogeneous culture during gradual weakening of neuronal connectivity. The weakening was achieved by progressive application of CNQX The pharmacological protocols described below were used identically in clustered and homogeneous cultures.in vitro networks contain both excitatory and inhibitory connections. However, for sake of simplicity in the comparison between experiments and model, in the experiments at DIV The We left active inhibitory synapses for experiments at DIV In the studies of network resilience to the weakening of connectivity, we studied the decay in spontaneous activity as a result of the gradual application of 6-cyano-7-nitroquinoxaline-2,3-dione , an AMPA-glutamate receptor antagonists in excitatory neurons. For The acquired images  can be computed in several ways, for instance through the jackknife method, the bootstrap algorithm, or by using the Fisher transformation The rich-club analysis computes the degree-degree (or weight-weight) correlation distributions, and respect to a null case of non-correlated degrees (or weights). It allows us to reinforce the assortativity analysis presented before. Assortative mixing, in principle, will induce a rich-club effect that should be clearly detectable for a wide range of degrees (or weights). We will first introduce the formulation for the calculation of rich-club in weighted networks as presented in The rich-club score is calculated as follows: The term To calculate this ratio on our functional networks, we have to consider that the network is not only weighted but also directed. Therefore, we need to adapt the former formulation for the case of weighted directed networks. For this reason we will consider the in- and out-strength of each node, expressed as This formulation allows us to calculate the rich-club coefficient for weighted directed networks. Note that for an undirected network Click here for additional data file.Figure S2Variance  and culture properties.A The variance in vitro, indicating that young cultures display a slower dynamics (larger delay times and therefore larger variance) than mature cultures. B The variance A show standard deviation.(PDF)Click here for additional data file.Figure S3Sensitivity of the functional network construction to the cut-off times. The cut-off determines the end of a sequence and therefore its variation modifies the set of burst chosen. The cut-off is set to (PDF)Click here for additional data file.Figure S4Relation between the structural traits of the network and the functional connectivity.A Dependence of the weight of the functional connections on the width of physical connections between directly connected clusters. The sketch conceptually shows the comparison between structural and functional links. The plot represents the analysis of B The dependence of the node strength on cluster size shows no correlation, indicating that the functional connectivity cannot be assessed from the size of the clusters. Data is based on the analysis of C For the same clusters, this plot shows that the activity of a cluster is independent of its size. D Activity within a burst is always initiated by a particular cluster, which triggers the sequential activation of all the downstream clusters. To quantify the importance of these ‘initiators of activity’ in network dynamics we computed the number of times that a cluster of a given size initiates a sequence of activations. The plot shows that there is no a significant correlation between initiation and size. The analysis is based on the study of (PDF)Click here for additional data file.Figure S5‘Rich-club’ analysis. The evaluation of the rich-club (PDF)Click here for additional data file.Figure S6Control experiment.A Bright field image of a clustered network whose spontaneous activity has been recorded for B Corresponding functional network. The size of the nodes is proportional to the size of the actual clusters, and their color is proportional to their strength. The weights of the links are both color and thickness coded. The darker the color, the higher the value of the observable. C Analysis of the 2 h recording in three blocks, (PDF)Click here for additional data file.Movie S1Representative recording in clustered networks. The movie shows the spontaneous activity in the network depicted in (AVI)Click here for additional data file.