Abnormal coactivation of leg extensors is often observed on the paretic side of stroke patients while they attempt to move. The mechanisms underlying this coactivation are not well understood. This study (1) compares the coactivation of leg extensors during static contractions in stroke and healthy individuals, and (2) assesses whether this coactivation is related to changes in intersegmental pathways between quadriceps and soleus (Sol) muscles after stroke.Thirteen stroke patients and ten healthy individuals participated in the study. Levels of coactivation of knee extensors and ankle extensors were measured in sitting position, during two tasks: maximal isometric voluntary contractions in knee extension and in plantarflexion. The early facilitation and later inhibition of soleus voluntary EMG evoked by femoral nerve stimulation were assessed in the paretic leg of stroke participants and in one leg of healthy participants.Coactivation levels of ankle extensors (mean ± SEM: 56 ± 7% of Sol EMG max) and of knee extensors  EMG max) during the knee extension and the ankle extension tasks respectively were significantly higher in the paretic leg of stroke participants than in healthy participants . Early heteronymous facilitation of Sol voluntary EMG in stroke participants (340 ± 62% of Sol unconditioned EMG) was significantly higher than in healthy participants (98 ± 34%). The later inhibition observed in all control participants was decreased in the paretic leg. Levels of coactivation of ankle extensors during the knee extension task were significantly correlated with both the increased facilitation (Pearson r = 0.59) and the reduced inhibition (r = 0.56) in the paretic leg. Measures of motor impairment were more consistently correlated with the levels of coactivation of biarticular muscles than those of monoarticular muscles.These results suggest that the heteronymous pathways linking quadriceps to soleus may participate in the abnormal coactivation of knee and ankle extensors on the paretic side of stroke patients. The motor impairment of the paretic leg is strongly associated with the abnormal coactivation of biarticular muscles. Stroke patients often present a pathological extension synergy in the affected leg while attempting to move voluntarily . This syAs part of this extension synergy, the coactivation of knee and ankle extensors may have a major effect on function. Since these anti-gravity muscles have a normal out-of-phase activation during gait ,6, theirIn patients with central nervous system lesions, abnormal synergistic coactivations have been mostly measured during isolated contractions in static conditions -12. In sSpinal interneurones are part of basic sensorimotor mechanisms that integrate descending and peripheral inputs. They can modulate the activity of motoneurones of muscles acting at the same joint or at different joints. Several studies have linked the malfunction of these pathways to motor deficits on the paretic side of stroke individuals. Modifications of the reciprocal inhibition of antagonist muscles acting at the same joint have been associated with changes in muscle tone , hyperreThis study aims (1) to compare the level of coactivation of knee and ankle extensors during maximal isometric voluntary contractions between stroke and healthy individuals; (2) to assess whether this coactivation is related to changes in the spinal pathways controlling heteronymous modulation of soleus activity by femoral nerve stimulation at the paretic leg. A preliminary report of the findings has been presented elsewhere .Thirteen stroke patients with chronic hemiparesis  and ten healthy individuals  of similar age (p = 0.5) participated in the study. All participants gave their written informed consent to the study, which had been approved by the internal ethics committee of the institutions of the Center for interdisciplinary research in rehabilitation of greater Montreal. Stroke participants were recruited based on the following inclusion criteria: a single cerebrovascular accident involving the motor cortex, internal capsule or sub-cortical areas as documented by brain imagery and resulting in motor deficits of abrupt onset affecting the contralateral leg. All patients tested had detectable patellar and Achilles tendon reflexes in the paretic leg. Moreover, all participants were able to produce sustained voluntary contractions of knee and ankle extensors, in order to perform the experimental tasks, which consisted of pushing on a pad with the leg and pressing on a fixed platform with the forefoot, in sitting position. Individuals with stroke were excluded if they were on antispastic, anxiolytic or antidepressant medication at the time of the study, or if they had receptive aphasia, hemispatial neglect, or passive range of motion limitation of the paretic leg that could interfere with the experimental positioning. Moreover, participants with stimulators (e.g. pacemaker) or metallic implants were excluded, as were those with orthopaedic or neurological disorders other than stroke.Prior to the experimental sessions, stroke participants were evaluated for level of motor coordination, level of motor impairment, degree of spasticity at the paretic leg and self-selected comfortable gait speed. The level of motor coordination of the paretic lower limb was measured using the Lower Extremity Motor Coordination Test (LEMOCOT), validated for stroke individuals . In thisA Biodex system 3 dynamometer  was used to measure the torques generated during two tasks: the maximal isometric knee extension and plantarflexion. Prior to each session, the dynamometer was calibrated. Figure ® Blue Sensor M) fixed in a bipolar configuration (2-cm center-to-center interelectrode distance leaving 8 mm spacing between the recording areas) over the belly of each recorded muscle. EMG signals were tested for crosstalk by performing standard muscle testing, rapid alternating movements and using the minimal interelectrode distance. EMG activities were collected using a telemetric system , relayed to a battery powered amplifier (2000x) with a bandwidth of 10 to 500 Hz and transmitted to a receiver interfaced with a PC card. These signals were acquired at a sampling rate of 1200 Hz using software constructed on a LabVIEW 5.0 platform  and stored on computer for later analysis.EMG activities of soleus (Sol), lateral gastrocnemius (GL), rectus femoris (RF) and vastus lateralis (VL) were simultaneously recorded. The skin was carefully prepared before placement of the disposable, self-adhesive, Ag/Ag-Cl surface electrodes  on the dynamometer. After the practice, a 5-minute rest period ensued. Before performing each experimental task, participants were first instructed to relax completely (background EMG below 5 μV). During the assessment, they were instructed to fold their arms across their chest and were verbally encouraged to reach their maximal isometric voluntary contraction as soon as possible after a "GO" signal and to hold a steady contraction for 4 s, with the visual feedback of the ongoing torque generated ,36. A mimax; GL at VLmax) and at RF . For the plantarflexion task, the coactivation levels of the quadriceps with reference to the plantarflexors were determined, for each trial, by the mean EMG activity of VL and RF within the 250-ms time windows when maximal mean EMG activity was reached at Sol  and at GL . For each muscle, the mean coactivation level was the average of the three trials per task and expressed as a percentage of the maximal EMG achieved in that muscle across the three trials of knee extension for VL and RF, and of plantarflexion for Sol and GL.All analyses were performed off-line. EMG signals were filtered using a zero-phase shift fourth-order digital Butterworth band-pass filter 20-125 Hz) and were full-wave rectified to obtain smoothed linear envelopes. The maximal torques in knee extension and plantarflexion were determined by averaging the maximal torque outputs of three trials for each task. Figure 0-125 Hz Participants were comfortably seated in a position similar to the one during the plantarflexion task of the assessment of coactivation in an adjustable reclining armchair with the foot strapped with Velcro to a fixed pedal. The femoral nerve was stimulated with a 1-ms duration monophasic rectangular pulse (Grass S88 stimulator) delivered by a cathode  at the femoral triangle and an anode (11.5 cm × 8 cm) placed at the postero-lateral aspect of the buttock. Stimulation intensity was progressively increased to determine the thresholds of the H reflex and of the M response (MT) for vastus lateralis. The intensity was then maintained at 2 × MT of vastus lateralis for the rest of the experiment. EMG activities of soleus and vastus lateralis were recorded  using bipolar surface electrodes  placed 2 cm apart (center-to-center). The recording electrodes were secured over the belly of vastus lateralis and soleus. EMG signals were first amplified (5000 x), then filtered (30-1000 Hz)  and finally, acquired at a sampling rate of 5 kHz. EMG signals were displayed on an oscilloscope and stored on computer for off-line analysis.max) of 5-second duration was first determined for each participant . All participants then produced isometric steady plantarflexions to activate soleus at 30% of EMGmax. Throughout the experiment, an analogue voltmeter facing the participant displayed visual feedback of the level of voluntary activity achieved at soleus (rectified and integrated EMG activity surface) for baseline control. Contractions had to be maintained for at least 3 s and a minimum rest period of 20 s was allowed between each of them. Random stimulations of femoral nerve at 2 × MT of vastus lateralis were performed during these contractions so that stimulation occurred in about one out of three contractions. The interval between the onset of soleus activation and the stimulation was also randomized. This ensured that participants would not be able to predict at which contractions the stimulation would be applied, or exactly when it would occur after the onset of soleus activation. For each leg tested, unconditioned and conditioned voluntary EMG activities of ten trials of femoral nerve stimulation were recorded during soleus voluntary contractions.Participants were instructed to press with the forefoot on the fixed platform in order to produce isometric plantarflexions. The level of EMG activity of soleus during maximal isometric voluntary contractions in plantarflexion . The later inhibition was assessed within 3 consecutive time windows of analysis of 12-ms duration each, from 12 to 24 ms, 24 to 36 ms and 36 to 48 ms after the zero central delay . Facilitation and inhibition levels were measured for each trial, at each time window, as the difference between the integrated rectified EMG after the conditioning stimulation (conditioned EMG) and before the stimulation (unconditioned EMG). This difference was expressed as a percentage of the control EMG measured within a window of 100 ms-duration just before the stimulation and then normalized for the duration of the time windows of analysis. Mean facilitation and inhibition of soleus voluntary EMG were assessed on ten trials of isometric contraction.For the coactivation assessment, Mann-Whitney U-tests were used to compare the levels of coactivation between the two groups . Wilcoxon signed rank tests were performed to compare the levels of coactivation of different muscles within the same group. Spearman rank correlations were used to correlate the scores obtained for clinical tests of coordination (LEMOCOT), motor recovery (CMSA), spasticity (CSI) and gait speed with the coactivation levels measured in stroke individuals. For the electrophysiological evaluation, analysis of variance (ANOVA) using Scheffe's method were performed in order to determine whether there was significant facilitation and inhibition throughout the windows of analysis before and after femoral nerve stimulation. Mann-Whitney U-tests were used to compare the levels of modulation between the two groups. Wilcoxon signed rank tests were performed to compare the levels of soleus EMG before and after femoral nerve stimulation within group or participant. Pearson correlations were used to correlate the levels of coactivation at the paretic leg with the levels of heteronymous modulation. The Spearman rank test was performed to assess the correlations between clinical scores  and electrophysiological data. P values ≤ 0.05 were considered significant. All statistical analyses were performed using the Statistical Package for Social Science (SPSS) software, version 17 for Windows.In both tasks tested, increased levels of coactivation were found in stroke participants compared to control participants. Figure Figure Figure An increase in the early facilitation and a decrease in the later inhibition of soleus voluntary EMG induced by femoral nerve stimulation was observed in stroke participants. Figure Figure max during the knee extension task were both correlated with the modulations within the first two time windows of analysis. No correlations were found between coactivation levels during the plantarflexion task and levels of heteronymous modulation in either stroke or control participants. Moreover, no correlations were found between coactivation levels and the last two time windows of analysis of the heteronymous modulation (24-36 ms and 36-48 ms).Table max, which was inversely correlated with comfortable gait speed. For the plantarflexion task, the amounts of coactivation of VL and RF were all inversely correlated with the coordination score. However, only the coactivations of RF were significantly correlated with level of motor recovery and degree of spasticity.The level of coordination of the paretic leg (LEMOCOT) was correlated with comfortable gait speed  and level of motor recovery (CMSA) , and tended to correlate with degree of spasticity (CSI) . Table The present study shows, in stroke individuals, an increased coactivation of knee and ankle extensors of the paretic leg during ankle and knee extensions, respectively. Only a few studies have quantified abnormal coactivations of leg extensors in stroke patients. An abnormal increased coactivation of knee and hip extensors has been found in standing position during maximal isometric extensions of the paretic knee and hip . Stroke One should point out that the effects of crosstalk could have influenced the levels of coactivation observed in the present study. However, these effects should be similar in stroke and control participants and thus cannot explain the difference in coactivation between the two groups. Moreover, we compared activity in a proximal  muscle versus a distal (soleus or gastrocnemius) limb muscle considered to be relatively far apart and recorded with the smallest interelectrode distance possible. This makes crosstalk less probable than comparing muscles that are close together in the same limb segment such as antagonist muscles.Our results showed coactivation levels of gastrocnemius lateralis in the paretic leg that were twice the levels found in healthy control subjects during knee extension. Similarly, coactivations in vastus lateralis were five times the levels found in control subjects during plantarflexion. These results concur with another study in which abnormal coactivations of gastrocnemius and rectus femoris were found during maximal knee extension and ankle extension after stroke, but in standing position .Our study also points out that, in some stroke individuals with severely impaired coordination, the activation of a muscle can be greater during its synergistic coactivation with other muscles than during its activation while attempting to produce maximal torque in a specific direction at which that muscle is normally preferentially activated. One should consider that the present study did not assess the ability of participants to produce isolated muscle activation. Participants were allowed to choose whatever strategy they want, including to produce coactivations, in order to perform the tasks. This allows us to assess which muscles would be preferentially recruited during these tasks. It should also be noted that the positioning and support provided during the two tasks could have favoured abnormal muscle recruitments and coactivations in both groups. Our results correspond with those of Neckel et al., (2006) showing that, for leg extension in stroke subjects, the secondary torque generated during the maximal voluntary contraction at another joint can be greater than the maximal voluntary torque generated at that joint. This concept of a greater recruitment of a muscle group during its synergistic coactivation than while attempting to specifically recruit it has been used in clinical rehabilitation in the Brunnstrom and proprioceptive neuromuscular facilitation approaches of stroke ,42.The mechanisms underlying these abnormal coactivations are not well understood. Weakness, changes in supraspinal influences and dysfunction of spinal pathways have been suggested as possible mechanisms contributing to the development of synergistic coactivations after stroke . There iAn increase in early heteronymous facilitation and a decrease in later inhibition of soleus voluntary EMG after femoral nerve stimulation were observed in the paretic leg of stroke individuals. Early heteronymous facilitation and later inhibition are thought to be mediated by intersegmental group Ia afferent excitation and recurrent inhibition from femoral nerve to soleus motoneurones, respectively ,38,46-48Increased coactivation of plantarflexors during the knee extension task was correlated with the enhanced heteronymous facilitation in the paretic leg. This suggests that changes of transmission in intersegmental pathways linking quadriceps to soleus could participate in the abnormal coactivation of ankle extensors when knee extensors are voluntarily activated. One can hypothesize that, in severely affected stroke patients, contractions of knee extensors at high levels produce an overall facilitative intersegmental influence on plantarflexors via short propriospinal pathways, leading to their abnormal coactivation. Conversely, no correlation was found between the increased coactivation of knee extensors during the plantarflexion task and the heteronymous modulation that we studied. This absence of correlation during plantarflexion strengthens the hypothesis that the intersegmental pathway that we stimulated is a propriospinal pathway specifically linking quadriceps afferents and soleus motoneurones. This also suggests that the increased coactivation of knee extensors during plantarflexion may involve other spinal pathways than those tested in the present study. These other pathways may modulate the activity of knee extensors by transmitting the influences from ankle extensors. Such intersegmental pathways do exist in humans, in whom quadriceps motoneurones receive both excitatory and inhibitory influences of group Ia afferents and recurrent inhibition from soleus, respectively ,51.This is the first time, to our knowledge, that changes in intersegmental pathways regulating the activity of motoneurones of muscles acting at different joints have been related to the impairment of selective muscular activation after stroke. An abnormal increase in the intersegmental facilitation of quadriceps activity by group I and group II afferents from common peroneal nerve was found at rest and during gait in stroke patients, but was not correlated to motor deficits -22. SomeCoactivation of plantarflexors during the knee extension task was correlated with self-selected gait speed in stroke individuals. The higher was the coactivation, the slower the gait speed. This is a new finding; it suggests that the inappropriate coactivation of leg extensors, as revealed in static conditions in the present study, may impede the accomplishment of a dynamic task such as gait. Knee and ankle extensors are both anti-gravity muscles that have a usual out-of-phase reciprocal activation during gait with quadriceps and calf muscles reaching their peak activation at the beginning and at the end of the stance phase, respectively ,6. PremaAnother finding of this study was the lower coactivation of biarticular muscles compared to the coactivation of monoarticular muscles in both sitting tasks on the Biodex, in healthy and stroke participants. This may reveal a differential control over mono- and biarticular muscles in this particular type of motor task, which requires torques to be produced in a specific direction at a single joint. Descending controls may favor the inhibition of biarticular muscles in these specific tasks since their activation would produce unwanted torques in other joints and thus reduce biomechanical efficacy.Furthermore, clinical measures were more correlated with the levels of coactivation of biarticular muscles than those of monoarticular muscles, in both motor tasks assessed. The lower was the motor function, the higher the level of coactivation of biarticular leg muscles in stroke participants. This suggests the importance of the control of biarticular muscles in leg function. Impairment of the specific control of biarticular muscles after stroke could contribute to motor deficits in hemiparesis, particularly in coordination of the lower limb. Several motor control studies have underlined the specific role of biarticular muscles in force orientation and energy distribution across joints in the lower limb ,60. ThisOnly changes in the intersegmental modulation from quadriceps to soleus (a monoarticular muscle) have been explored in the present study. However, changes in intersegmental modulation of biarticular muscles in the paretic leg have not been investigated. When considering the strong relationship between the coactivation of biarticular muscles and motor function found in our study, changes in spinal modulation of these muscles might be strongly related with motor function of the paretic leg. Such modulation involving spinal pathways liking quadriceps to gastrocnemius does exist in humans ,51. FutuThere is an abnormal coactivation of knee and ankle extensors during maximal static contractions of the paretic leg of stroke patients with coordination deficits. Alterations of intersegmental pathways linking quadriceps to soleus are specifically correlated with the increased coactivation of ankle extensors during the knee extension task. Thus, these propriospinal pathways may participate in abnormal coactivations of ankle extensors while knee extensors are voluntarily activated in the paretic leg. Coactivations of biarticular muscles during static tasks directed at a single joint appear to be more related to motor deficits after stroke, and thus may have more functional impacts than coactivations of monoarticular muscles. Future studies should investigate the role of these and other intersegmental pathways in the motor deficits observed during dynamic conditions such as gait after stroke and in other central nervous system lesions.The authors declare that they have no competing interests.JOD prepared subjects, carried out the experiments, collected and analyzed the data, and drafted the manuscript. EM prepared subjects, carried out the experiments and collected the data. SAM analyzed the data and helped draft the manuscript. DB designed the experiments and helped draft the manuscript. RF designed the experiments, analyzed the data and helped draft the manuscript. All authors read, edited, and approved the final manuscript.


Drosophila, hunchback (hb) and knirps (kni). Our model includes not only mutual repression between hb and kni, but also the stochastic and cooperative activation of hb by the anterior morphogen Bicoid (Bcd) and of kni by the posterior morphogen Caudal (Cad), as well as the diffusion of Hb and Kni between neighboring nuclei. Our analysis reveals that mutual repression can markedly increase the steepness and precision of the gap gene expression boundaries. In contrast to other mechanisms such as spatial averaging and cooperative gene activation, mutual repression thus allows for gene-expression boundaries that are both steep and precise. Moreover, mutual repression dramatically enhances their robustness against embryo-to-embryo variations in the morphogen levels. Finally, our simulations reveal that diffusion of the gap proteins plays a critical role not only in reducing the width of the gap gene expression boundaries via the mechanism of spatial averaging, but also in repairing patterning errors that could arise because of the bistability induced by mutual repression.Embryonic development is driven by spatial patterns of gene expression that determine the fate of each cell in the embryo. While gene expression is often highly erratic, embryonic development is usually exceedingly precise. In particular, gene expression boundaries are robust not only against intra-embryonic fluctuations such as noise in gene expression and protein diffusion, but also against embryo-to-embryo variations in the morphogen gradients, which provide positional information to the differentiating cells. How development is robust against intra- and inter-embryonic variations is not understood. A common motif in the gene regulation networks that control embryonic development is mutual repression between pairs of genes. To assess the role of mutual repression in the robust formation of gene expression patterns, we have performed large-scale stochastic simulations of a minimal model of two mutually repressing gap genes in  Drosophila, we show that mutual repression can dramatically enhance both the steepness and precision of the boundaries of the gene-expression domains. Moreover, it can buffer against variations in the protein (morphogen) gradients that provide the positional information to the developing embryo. Lastly, our simulations show that diffusion of the expressed protein not only sharpens the boundaries of the expression domains, but also can repair defects that are formed within these domains.Embryonic development is controlled by spatial patterns of gene expression that seal the fate of each cell in the embryo. However, while development is typically very precise, gene expression is often very noisy. Indeed, a key question in current biology is how embryonic development can be so precise, while the underlying processes are very erratic. To address this question, we have performed extensive stochastic simulations of a canonical motif in the gene regulation networks that drive embryonic development, namely mutual repression between pairs of genes. By studying a minimal model of two mutually repressing genes in the fruitfly  DrosophilaDrosophila melanogaster. Our results show that mutual repression between target genes can markedly enhance both the steepness and the precision of gene-expression boundaries. Furthermore, it makes them robust against embryo-to-embryo variations in the morphogen gradients.The development of multicellular organisms requires spatially controlled cell differentiation. The positional information for the differentiating cells is typically provided by spatial concentration gradients of morphogen proteins. In the classical picture of morphogen-directed patterning, cells translate the morphogen concentration into spatial gene-expression domains via simple threshold-dependent readouts Drosophila melanogaster is arguably the paradigm of morphogenesis. During the first 90 minutes after fertilization it is a syncytium, consisting of a cytoplasm that contains rapidly diving nuclei, which are not yet encapsulated by cellular membranes. Around cell cycle 10 the nuclei migrate towards the cortex of the embryo and settle there to read out the concentration gradient of the morphogen protein Bicoid (Bcd), which forms from the anterior pole after fertilization hunchback (hb), which is expressed in the anterior half of the embryo. In spite of noise in gene expression, the midembryo boundary of the hb expression domain is astonishingly sharp. By cell cycle 11, the hb mRNA boundary varies by about one nuclear spacing only et al. revealed that the Hb concentrations in neighboring nuclei exhibit correlations and the authors suggested that this implies a form of spatial averaging that enhances the precision of the posterior Hb boundary The fruit fly hb, but a number of gap genes, and pairs of gap genes tend to repress each other mutually. Interestingly, repression between directly neighboring gap genes is weak, whereas repression between non-adjacent genes is strong hb forms a strongly repressive pair with knirps (kni) which is expressed further towards the posterior pole; both genes play a prominent role in the later positioning of downstream pair-rule gene stripes Bcd induces the expression of not only hb and kni. Our model includes the stochastic and cooperative activation of hb by Bcd and of kni by the posterior morphogen Caudal (Cad) e.g., the interactions of hb and kni with krüppel (kr) and giant (gt) While the role of antagonistic interactions in the formation of gene-expression patterns has been studied using mean-field models One of the key findings of our analysis is that mutual repression enhances the robustness of the gene expression domains against intra-embryonic fluctuations arising from the intrinsic stochasticity of biochemical reactions. Specifically, mutual repression increases the precision of gene-expression boundaries: it reduces the variation hb and kni. Moreover, mutual repression can strongly enhance the steepness The simulations reveal, perhaps surprisingly, that mutual repression hardly affects the noise hb and kni respectively overlap, bistability can arise in the overlap zone. Yet, the mean waiting time for switching is longer than the lifetime of the morphogen gradients, which means that the hb and kni expression patterns are stable on the relevant developmental time scales. This also means, however, that when errors are formed during development, these cannot be repaired. Here, our simulations reveal another important role for diffusion: without diffusion a spotty phenotype emerges in which the nuclei in the overlap zone randomly express either Hb or Kni; diffusion can anneal these patterning defects, leading to well-defined expression domains of Hb and Kni. Finally, we also study a scenario where hb and kni are activated by Bcd only. While this scheme is not robust against embryo-to-embryo variations in the morphogen levels, mutual repression does enhance boundary precision and steepness also in this scenario.Our results also show that mutual repression can strongly buffer against embryo-to-embryo variations in the morphogen levels by suppressing boundary shifts via a mechanism that is akin to that of hb and kni expression can be detected We consider the embryo in the syncytial blastoderm stage at late cell cycle 14, ca. hb and kni by the morphogens Bcd and Cad, respectively, and mutual repression between hb and kni  of Gillespie Three key characteristics of gene expression boundaries are 1) the noise in the protein concentration at the boundary; 2) the steepness of the boundary; 3) the width of the boundary. While these quantities may make intuitive sense, their definitions are not unambiguous. Equally important, different definitions will reveal different properties of the system.Let's consider the variance in the copy number Fig. S2 in Now that we have characterized the fluctuations in the copy number and the boundary position, the next question is how fluctuations in the copy number affect the steepness of the boundary. In particular, a gene-expression boundary can be shallow either because at each moment in time the interface is shallow, or because at each moment in time the interface is sharp yet the interface fluctuates in time, leading to a smooth profile. The question is thus how much the gradient of the mean concentration profile, In the rest of the manuscript, we will predominantly focus on the properties of individual embryos, and average quantities are typically averages over time and the circumference. For brevity, therefore, hb and kni promoters have 5 copies of their respective morphogens bound. Without mutual repression, thus when Hb and Kni cannot bind to their respective target promoters, these profiles describe the probability that hb and kni are activated by their respective morphogens. Indeed, without mutual repression and without Hb and Kni diffusion, the Hb and Kni concentration profiles would be proportional to their respective morphogen-activation profiles hb promoter is induced, meaning that it has 5 copies of Bcd bound to it and no Kni, and the profile of the likelihood hb is activated by Bcd, yet repressed by Kni, in which case hb is not expressed. It is seen that repression by kni almost fully inhibits hb expression beyond the half-activation point, where hb would be expressed without kni repression (see inset Panel A). Indeed, mutual repression effectively cuts off protein production beyond midembryo. The production probability therefore changes more abruptly along the AP axis, leading to a higher steepness of the protein profiles near midembryo. For In steepens the effective gene-activation profile et al. in cell cycle 14 Both with and without Hb - Kni mutual repression the trade-off between noise and steepness reduction leads to an optimal diffusion constant mean gene-activation profiles increasesminimalA key parameter controlling the precision of the gap-gene expression boundaries, is the degree of cooperativity by which the gap genes are activated by their respective morphogens—this determines the profile steepness of the average gap-gene promoter activity. To investigate this, we have lowered the effective Hill coefficient from its baseline value of 5 by reducing the number As a standard we assume very tight binding of the Hb and Kni dimers, “the repressors”, to their respective promoters. To test how this assumption affects our results we performed simulations in which we systematically varied the repressor-promoter dissociation rate Since the precise gap protein expression level is not known, we also varied the maximal protein copy number ate  see . Fig. S9see also . The scahb. Indeed, in vivo the Hb profile is shaped by other forces, like mutual repression. In the kni - kr double mutant, the Hb boundary at midembryo shifts posteriorly hb and kni are half-maximally activated by their respective morphogens, Bcd and Cad. While the threshold positions could be varied by changing the threshold morphogen concentrations for half-maximal gap-gene activation (for example by changing the morphogen-promoter dissociation rates), we will vary these positions by changing the amplitude of the morphogen profiles by a factor Although the Bcd copy number at midembryo has been determined experimentally hb and kni are half-maximally activated by their respective morphogens coincide at midembryo, meaning that the domains in which hb and kni are activated beyond half-maximum are adjoining, but do not overlap—this is the scenario discussed in the previous sections. When hb is half-maximally activated by its morphogen is shifted posteriorly, while that of kni is shifted anteriorly, creating an overlap between the two regions where hb and kni are activated. In this “double-activated region” both hb and kni are activated by their respective morphogens, yet they also mutually repress each other. This may lead to bistability. To probe whether this is the case, we performed a bifurcation analysis of the mean-field chemical-rate equations of isolated nuclei, implying that hb -dominant state is more stable than the kni -dominant state, and vice versa. The emergence of such a “spatial switch” along the AP axis is also captured by our mean-field, bifurcation analysis  is essentially fully expressed. For hb is highly expressed . For hb and kni simultatenously repress each other); instead, the hb and kni domains formed near the poles slowly invade the overlap region  the boundary does not move beyond the position for hen  see . While tThe fact that mutual repression can pin expression boundaries, dramatically enhances the robustness against embryo-to-embryo variations in the morphogen levels. We did not sample inter-embryo variations in hb - kni interface. Moreover, overlapping morphogen activation domains decrease the steepness of the expression boundaries (panel A), and this increases the boundary width While mutual repression proves beneficial in buffering against embryo-to-embryo variations in morphogen levels, the question arises whether overlapping morphogen-activation domains does not impair robustness to intrinsic fluctuations arising from noisy gene expression and diffusion of gap gene proteins. We found that this depends on the Hill coefficient of gap-gene activation, which depends on the number Since correlated upregulation of both morphogen levels is a special case, we also studied the effect of uncorrelated activator scaling. To this end, only the Bcd level was multiplied by a global factor hb expression boundary shifts posteriorly with increasing bcd gene copies. It is seen that the agreement between simulation and experiment is very good: while To estimate the boundary variance due to inter-embryo variations in morphogen levels, we fitted a generic logarithmic function The output noise at the Hb boundary remains largely unaffected  gap gene patterns form in the absence of Bcd In the following we describe details of our parameter choice and sampling technique. To unravel the mechanisms by which mutual repression shapes gene-expression patterns, it is useful to take the Cad - Kni -system to be a symmetric copy of the Bcd - Hb -system. Cad thus inherits its parameters from Bcd and Kni from Hb, if not otherwise stated. Table S2 in We assume all promoter binding rates to be diffusion limited and calculate them via hb and kni by their respective morphogens, the morphogen-promoter dissociation rate is given by hb and kni (the repressor-promoter unbinding rate) hb and kni are fully activated by their respective morphogens . This requires tight repression, yielding dissociation constants To model cooperative activation of The parameters of the exponential morphogen gradients are chosen such that the number of morphogen molecules at midembryo and the decay length of the gradient are close to the experimentally observed values for Bcd, 690 and The copy numbers of both monomers and dimers and the effective gap gene degradation rate ions see . Based oOne of the key parameters that we vary systematically is the internuclear gap gene diffusion constant hb and kni expression boundaries.All simulations are split into a relaxation and a measurement run. During the relaxation run we propagate the system towards the steady state without data collection. To reach steady state, as a standard we run lts for   form, hoIn steady state, we record for each row of nuclei and with a measurement interval of Error bars for a given quantity are estimated from the standard deviation among Text S1Supporting information. More detailed information on parameter choice, data measurement and analytical estimates and results on the system with altered cooperativity, the systems with altered production dynamics and the systems where both gap genes are activated by the same morphogen gradient.(PDF)Click here for additional data file.Video S1Establishment of gap gene expression patterns for a low diffusion constant of the gap proteins. Movie of the total concentration of Hb as a function of time for hb and kni are activated by their respective morphogens, Bcd and Cad. These crystallites then coarsen and join the Hb domain formed near the anterior pole. The green line marks the positions where the total Hb concentration crosses the boundary threshold value.(MP4)Click here for additional data file.Video S2Establishment of gap gene expression patterns for a low diffusion constant of the gap proteins. Movie of exactly the same system trajectory as in Video S1, only now showing the difference between the total Hb and total Kni copy number.(MP4)Click here for additional data file.Video S3Establishment of gap gene expression patterns for a high diffusion constant of the gap proteins. Movie of the total concentration of Hb as a function of time for (MP4)Click here for additional data file.Video S4Establishment of gap gene expression patterns for a high diffusion constant of the gap proteins. Movie of exactly the same system trajectory as in (MP4)Click here for additional data file.


Background: Modern techniques for breast-reduction surgery depend on large surface de-epithelialization. The current gold standard technique involves the use of a scalpel for sharp debridement and is a time-consuming process that is labor-intensive and often requires an assistant to stabilize the breast. Surgeons who perform breast-reduction surgery continue to search for instruments and innovations that may reduce the time and effort required for successful de-epithelialization. One such innovation is the use of the VersaJet Hydrosurgery system. The purpose of this article is to highlight an important complication that may result from the use of this device. Methods: The authors describe their experience with the VersaJet Hydrosurgery System in reduction mammoplasty of 28 breasts. Results: The authors experienced several complications characterized by the formation of epithelial inclusion cysts requiring reoperation. Conclusion: The VersaJet Hydrosurgery system may lead to quicker speeds of de-epithelialization as compared with traditional methods but poses a risk of epidermal cyst formation. Although many surgeons successfully use the traditional scalpel technique, many others have applied novel strategies and equipment to hasten this process with mixed results. For instance, some surgeons report an increased speed of de-epithelialization when using the dermatome. However, this method is prone to inconsistent results related to tissue skipping.1,Our division experienced favorable outcomes while using the VersaJet Hydrosurgery System for tissue debridement in the setting of pressure ulcers and burn wounds. This instrument allowed expeditious debridement of large areas of granulation tissue in ulcerative wounds to prepare for skin grafting. Our results in this context were positive and consistent with the successful outcomes reported in the literature.3The VersaJet technique allowed for what appeared to be uniform de-epithelialization in a single plane of tissue. Unlike with the use of a blade, there was no dulling; thus, the operation proceeded uninterrupted and the de-epithelialization phase lasted approximately 10 minutes. Despite these benefits, in our sample size of 14 patients, 9 patients experienced no negative outcomes while 5 patients experienced serious complications. In contrast to the results of the Lonergan and Moquin study of 20 patients, which revealed no complications,-de-epithelialization was a suboptimal descriptor for VersaJet Hydrodissection of the breast pedicle.A review of the literature yields only a few articles discussing the depth required to properly de-epithelialize. Older reports of dermal pedicle biopsies indicate that standard “hand and knife” techniques remove the epidermis and a variable amount of dermis.6The VersaJet Hydrodissector, while offering increased speeds of de-epithelialization over traditional methods, presents a risk of epidermal cyst formation that cannot be ignored. Although we are enthusiastic about the potential uses for this machine, we feel the need to highlight our complications to the plastic surgery community. Further studies will be required to investigate the histological changes in the breasts with epidermal cyst formation, and an investigation to determine the depth of de-epithelialization afforded by the VersaJet system. Perhaps future devices may use the fundamentals behind the hydrodissector to create a safe de-epithelialization device for breast-reduction surgery. Breast surgeons performing breast-reduction surgical procedures should be cautious before implementing this technology.


Karate tournaments consist of two equally important karate disciplines: the kumite and kata competitions. Due to being based both on the distinctive selection of movement techniques and their kinematic and kinetic patterns, we hypothesized that the elite kumite and kata competitors could differ regarding their anthropometric and physical performance profiles. Thirty-one senior male karate competitors of the national karate team  participated in this study. The tests applied included both the assessment of anthropometric  and the following physical performance measurements: the adductor and hamstring flexibility (sideward leg splits test), speed and acceleration (20-m sprint test with 10-m acceleration time), explosive power (countermovement and standing triple jump), agility (“T”- test) and aerobic endurance (20-m multistage shuttle run test). The kumite competitors revealed a larger body size through body height (p = 0.01) and mass (p = 0.03), while the differences in body composition were non-significant. The kumite competitors also demonstrated higher acceleration (p = 0.03) and explosive power . A 6-7° higher flexibility of the kata competitors remained somewhat below the level of significance (p = 0.09). The findings could be interpreted by the distinctive differences in the movement techniques. Specifically, a higher explosive power could be beneficial for kumite, while both a smaller stature and higher flexibility (particularly of the lower extremity) could be important for the exceptionally low postures of the kata competitors. Although further elucidation is apparently needed, the obtained finding could be of importance for both the early selection and training of karate competitors. Karate is arguably one of the most popular martial arts practiced worldwide. Traditional karate training involves basic techniques, kata, and sparring activities . Modern In past, the demands of karate competitions used to be similar for both disciplines, as well as for the standard training programs. As a consequence, the participants often used to successfully compete in both the kata and kumite. However, the alternation of the competition rules  has made the kumite competitions both more dynamic and attractive . Unlike In general, the selection of athletes should be primarily based on the abilities and skills that have a crucial influence on sport performance, where genetic factors could be of considerable importance . Taking The literature reviewed above suggests that despite the general importance of physical abilities and anthropometric characteristic for the purpose of both, sport selection and evaluation of the training process , there iThirty-one elite senior male members of the national karate team participated in this study. Nineteen of them were medalists at the junior (16–18 years of age) and senior World, European and Balkan championships. Nineteen of them were kumite, while 12 were kata competitors . None ofThe present study is an experimental one based on a cross-sectional approach. We particularly focused on those characteristics that could differ between two groups regarding the requirements of their respective discipline-specific movements .Subjects were instructed to avoid any strenuous physical activity two days prior to the experiment to minimize the possible effect of fatigue. The experiment was carried out within a single testing session. It included anthropometric measurements and medical screening, followed by physical performance testing.2). Anthropometric measurements were taken by the same experimenter according to standard procedures.Body height (BH) and body mass (BM) were measured to the nearest 0.5 cm and 100 g, respectively. Thereafter, the body mass index was assessed  was selected for direct assessment of the flexibility of lower extremity. It predominantly assesses the flexibility of the hamstring muscles of the front leg and the adductor muscles of the back leg  and maximum speed over a short distance (10 m flying start – 10FS). Although the movement is not karate-specific, the test selection was based on the presumed importance of rapid movement initiation and speed for elite kumite competitors (petitors . The subThe T-test (TT) was selected for the assessment of agility. It has been recently reported that the ability to change direction could be of high importance for success in martial arts  and Standing triple jump (STJ) were used for indirect assessment of explosive power of leg extensors in the vertical and horizontal plane. The tests were selected because of their presumed validity for the assessment of performance of lower limbs in karate competitors (petitors . It coulpetitors .2g (g = 9.81 m/s2). A high intra-trial and test-retest reliability of CMJ have been reported (ICC>0.9)  that records flight time (t). The rise of the center of gravity above the ground (height in meters) was measured from time of flight  applying the ballistic law: h = ⅛ tICC>0.9) .STJ - The subjects were instructed to jump as far as possible performing a standing triple jump from a standard standing position. The distance from the starting point to the landing point at the heel contact was used for further analysis. The precision of the measurement was 1 cm. 20-m Shuttle run test (SR) was used for the assesment of aerobic endurance. With the exception of the scoring system, the original test protocol of the multistage Shuttle run test was followed (2). Note that followed . In shorfollowed , we scorp = 0.05. All statistical tests were performed using SPSS 16.0 .Standard descriptive statistics (mean and standard deviation) were calculated for each variable. Significant statistical differences between the two groups were tested by means of the two-tailed, independent t-test. Normality of distribution of residuals was tested by means of the Kolmogorov-Smirnov test. Statistical significance was set at Overall, the data suggest a larger body size of the kumite athletes, while the difference in body composition as assessed by BMI was not statistically significant .The Kolmogorov-Smirnov test revealed violation of normal distribution in none of the tested variables . The kumWithin the present study we aimed to test the anthropometric and physical performance profiles of elite karate kumite and kata athletes in order to identify the differences that could be of importance for future selection, training and testing of karate competitors. The findings were mainly in line with our hypotheses based on the differences in both the selection and the patterns of typical techniques performed by kumite and kata competitors.The recorded anthropometric variables  of the tested karate athletes were comparable with those of elite karate athletes tested in earlier studies .However, it should be kept in mind that both the range and distribution of body size of the kumite competitors tested in our study were inevitably affected by the distribution of weight categories prescribed by the competition rules. Nevertheless, presuming that weight categories correspond to body size distribution of the general population of kumite competitors , our results revealed a higher body size in kumite than in kata competitors. Part of explanation could be found in the effects of body size scale that selectively affect the different physical performance c.f., . SpecifiRegarding physical performance, we found a higher ability to accelerate the whole body (as assessed by both the 10S and STJ test) in the kumite competitors. Again, the obtained difference could be explained by the specific requirements of the typical kata and kumite competitions. Specifically, a success of both the attacking and defensive kumite techniques highly depends on the ability to rapidly initiate the change of body position in horizontal direction. The kata competitors, however, are expected to demonstrate an excellence in kinematic patterns of the prescribed techniques performed at a moderate pace what does not require rapid changes in body position.Regarding the remaining tests of maximum performance, one could speculate that a lack of differences in CMJ between two groups could be a consequence of a need of the kumite competitors to rapidly change the body position in horizontal, but not in vertical direction. Note that only a moderate relationship (i.e. the correlation coefficients between 0.46 and 0.77) were found between the abilities to accelerate in vertical  and horizontal direction (i.e. sprinting) , while aWe did not find differences between the two groups regarding aerobic endurance (SR), agility (TT) and flexibility (SdLS). The former finding could be explained by similar aerobic demands of the kata and kumite during both training and competition or, alternatively, by a relatively low sensitivity of the applied test . TherefoTo conclude, the findings suggest that kumite competitors could be of larger body size, higher ability for initiation of body movement in horizontal direction and, possibly, relatively reduced flexibility of lower limbs. Since only elite competitors were tested, the findings could be of importance for both the selection and training design of karate athletes. Future elucidation of the discipline specific profiles of the kumite and kata competitors could include larger numbers of the anthropometric measures, physical performance and skill-specific tests, as well as the extension of the same research to female competitors. However, based on the essential differences in the skills required from elite kumite and kata competitors , a particularly promising extension of this line of research could be towards more neurophysiologicaly oriented tests, such as the reaction time and the ability for a rapid stimulus-reaction processing.


Both neuropsychological and functional neuroimaging studies have identified that the posterior parietal lobe (PPL) is critical for the attention function. However, the unique role of distinct parietal cortical subregions and their underlying white matter (WM) remains in question. In this study, we collected both magnetic resonance imaging and diffusion tensor imaging (DTI) data in normal participants, and evaluated their attention performance using attention network test (ANT), which could isolate three different attention components: alerting, orienting and executive control. Cortical thickness, surface area and DTI parameters were extracted from predefined PPL subregions and correlated with behavioural performance. Tract-based spatial statistics (TBSS) was used for the voxel-wise statistical analysis. Results indicated structure-behaviour relationships on multiple levels. First, a link between the cortical thickness and WM integrity of the right inferior parietal regions and orienting performance was observed. Specifically, probabilistic tractography demonstrated that the integrity of WM connectivity between the bilateral inferior parietal lobules mediated the orienting performance. Second, the scores of executive control were significantly associated with the WM diffusion metrics of the right supramarginal gyrus. Finally, TBSS analysis revealed that alerting performance was significant correlated with the fractional anisotropy of local WM connecting the right thalamus and supplementary motor area. We conclude that distinct areas and features within PPL are associated with different components of attention. These findings could yield a more complete understanding of the nature of the PPL contribution to visuospatial attention. Attention refers to both the preparedness for and selection of certain environmental or mental aspects In order to isolate the functional components of attention and to investigate their association, Fan and colleagues The posterior parietal lobe (PPL) has long been recognized as a neural substrate of visuospatial attention. With its extensive connectivity to cortical and subcortical regions in occipital However, the roles of distinct parietal subregions on attention function are still highly debated, mainly due to the inconsistency among the attention models and the variations in the utilized imaging modalities. For example, alerting sometimes is termed as sustained attention or vigilance. Although there are slight differences between them, these functions are considered as part of a system controlling the intensity of attention, rather than its selectivity On the other hand, orienting represents the characteristic of selective attention. Data from functional imaging studies manifested that bottom-up orienting, mediated by stimulus salience and/or relevance, was subserved by inferior parietal regions Finally, executive control is commonly measured using tasks in which there is an incompatibility between dimensions of the stimulus or response. Several recent studies have suggested that the temporoparietal junction in the right hemisphere responds to intersensory and sensorimotor conflict Generally, it is proposed that human PPL is involved in three distinct cognitive functions: spatial perception, vision-for-action and visuospatial attention Healthy Chinese participants with ages ranging from 17 to 20 years were recruited from the local community. There were four inclusion criteria: (1) The participants were right-handed investigated by Edinburgh inventory A version of the ANT devised by Fan and colleagues 2) and 3 non-diffusion-weighted images (b = 0 s/mm2). From each participant 56 axial slices were acquired and the diffusion sequence was repeated 2 times to increase signal-to-noise ratio.DTI was carried out using a 3-T GE Signa scanner . The images were collected using diffusion-weighted imaging with an array spatial sensitivity encoding technique (ASSET) based on a single-shot echo-planar imaging sequence . The DTI scheme included the collection of 30 directions with non-collinear diffusion gradients  pulse sequence with 174 slices  was used to acquire the anatomical images for structural analyses.Firstly the total accuracy of each subject was calculated and the trials with incorrect responses or with RTs longer than 1500 ms or shorter than 200 ms were excluded to avoid the influence of the outliers. We also removed responses following erroneous ones to avoid post-error slowing effect. Since RTs were not normally distributed, we used median RT per condition as raw scores. Finally, instead of the conventional subtraction measure The MR images were first processed with the CIVET MRI analysis pipeline (version 1.1.9) developed at Montreal Neurological Institute (MNI) to automatically extract and co-register the cortical surfaces for each subject. The main pipeline processing steps included: (1) The native three-dimentional structural MR image of each subject was corrected for non-uniformity using the N3 algorithm In this study, we employed the automated anatomical labelling (AAL) template The DTI data was pre-processed using FSL . Firstly the diffusion data were corrected for eddy currents and head motion and the two acquisitions were averaged. The averaged images were masked to remove skull and non-brain tissue using the FSL Brain Extraction Tool (BET) We then used tract-based spatial statistics (TBSS) 1-weighted structural image of MDT was first co-registered to the resampled b0 image in MNI space using a linear transformation. The transformed structural image was then mapped to the MNI single-subject MRI brain template using a nonlinear transformation. The resulting inverse transformation was then used to warp the AAL mask from the brain template to the “skeleton” brain in which the discrete labeling values were preserved by using a nearest neighbor interpolation method Finally, we also used AAL template to segment the WM skeleton. To do this, TVoxels identified in the TBSS analysis were then used as seed masks for multi-fiber probabilistic tractography The frequency histograms and Shapiro-Wilk tests showed that the behavioural data presented approximately normal distribution. To explore the relationships between the different ANT components, we correlated each of the ratio scores after partialling out gender and age. We then tested for main effects of gender on ANT scores and AAL-based variables using independent samples t-test. Linear relationships with ANT scores were investigated for all measures of the ten PPL labels  in order to simplify comparisons across measures and regions.In order to explore the unique contributions of the different measures, stepwise multiple regressions with each of the ANT scores as the dependent variable, age, gender and brain volume as confounding covariates, and regional cortical thickness, surface area, and FA as predictors were performed. The same procedure was repeated for MD instead of FA. Separate analyses were performed using FA and MD to avoid underestimates of contributions of these parameters because FA and MD are indexes reflecting the same diffusion eigenvalues The accuracy of ANT performance was 95.3%±3.4%, indicating that the participants understood the instructions and were able to respond reliably. There were no correlations with ANT scores on overall accuracy before and after partialling out gender and age. Therefore, the accuracy was not included as covariate in the subsequent analyses. The ratio scores of alerting, orienting and executive control effects as well as their correlations were summarized in 2, p<0.001) and brain volume  as compared to the females. Subsequent AAL-based analyses indicated that the cortical thickness of the left SMG, and the surface area of the right IPL, bilateral SPL and precuneus in men was significantly larger (p<0.005) than in women. Interhemispheric differences were not investigated here because it might be confounded by the asymmetry of AAL partitions between hemispheres.Generally, males showed larger global mean cortical thickness , surface area . We also found a correlation between orienting score and cortical thickness of both the left  and right  SPL, but both failed to reach the statistical significance of 0.005. No significant findings emerged between cortical parameters and alerting as well as executive control performance (p>0.05).AAL-based correlation analyses revealed a significant negative correlation between orienting score and MD of WM skeleton underlying the right IPL , after controlled for age and gender. A positive relationship was also found between executive control performance and skeleton MD  as well as RD  in the right SMG. Nevertheless, no significant correlations were found between ANT scores and skeleton FA underlying the PPL (p>0.005).Voxel-wise analysis indicated that alerting scores were associated with regional skeleton FA underlying the right frontal lobe  . No sign2 = 0.61) included cortical thickness of the right IPL  and AG , as well as local FA underlying the right IPL ; for MD, the optimal model  also included cortical thickness of the right IPL  and AG , as well as local MD underlying the right IPL . The similar multiple regression analyses for alerting and executive control were not conducted because of the lack of significant relationships to the cortical thickness and surface area during the aforementioned correlation analyses.The results from the stepwise multiple regressions on orienting performance verified the above findings. Specially, for FA as one of the variables, the optimal model , instead of 50 samples. Then the individual tracts were binarized and summed to produce group probability map . The resPartial correlations between cortical thickness and other measures when controlled for age, gender and brain volume were shown in The present study demonstrated that interindividual variations in the ability of spatial attention reflect variability in cortical thickness and WM integrity in distinct posterior parietal subregions. Specifically, the orienting performance was linked to the cortical thickness of the right IPL and AG, and the interhemispheric WM connections between the bilateral IPLs. Additionally, the executive control scores were significantly associated with the MD/RD of the WM underlying the right SMG. We found no significant correlations for parietal structures with the alerting effect. Instead, current study indicated that alerting may be partly mediated by the structural connectivity between the right supplementary motor area and the ipsilateral thalamus.Our main finding was that variations of the orienting performance were associated with the cortical thickness and FA values of the right IPL in the multiple regression analysis. One caution is that IPL in AAL template occupies the anterio-inferior part of the lateral intraparietal sulcus adjacent to but not composed of the SMG and AG. Although the intraparietal sulcus has been designated as a crucial node of dorsal attention network in Corbetta’s dichotomy model It has been proposed that a dynamic balance exists between the two hemispheres in orienting attention It is not surprising that we also detected an association between cortical thickness of the right AG and orienting performance. Using TMS on the bilateral AG and SMG, one study had found that only the right AG mediated spatial orienting during two discrete time periods after target onset For executive control, we also found significant correlations between this component and MD/RD of the WM underlying the right SMG after correcting for multiple comparisons. Executive control is construed as the monitoring and resolution of conflict in decision making, error detection or regulation of thoughts and feelings Notably, the TBSS statistical analysis was applied to the whole brain skeleton to localize any area of significant association with each of the three attention components without any priori hypothesis. The analysis revealed that alerting performance was significantly correlated with the FA of local WM connecting the right thalamus and supplementary motor area through the posterior limb of internal capsule. The alerting component during ANT task, which is also defined as phasic or exogenous alertness The intrathalamic nuclei are known to be engaged in maintaining a state of high vigilance or intrinsic alertness  In addition to examining the brain structural substrates for attention function, we investigated the associations between cortical morphological properties and WM parameters. Compared to the surface area, cortical thickness was more strongly related to the WM parameters, especially the FA and RD. In line with one existing study Besides, we also revealed that cortical thickness correlated negatively with surface area within some PPL subregions, indicating that individuals with thinner cortex tend to have larger surface area There are some limitations of the applied techniques that need to be addressed. First, although the AAL template had been used to define the network nodes in the previous cortical The current study evaluated MRI and DTI parameters in PPL respectively, and determined their relationships with the alerting, orienting and executive control performance of ANT in healthy individuals. The most significant novelty is the validation of the distinct roles of PPL subregions on the three sub-networks of attention. We found that orienting function was associated with the cortical thickness of the IPL and AG, and the FA underlying the right IPL. Further probabilistic tractography demonstrated that the interhemispheric WM integrity between the bilateral IPLs mediated the orienting performance. In addition, we revealed that the executive control scores were significantly associated with MD/RD of WM underlying the right SMG. Another notable finding is that alerting performance was linked to FA of local WM connecting the right thalamus and supplementary motor area through the posterior limb of internal capsule. In accordance with previous functional imaging and lesion studies, our findings will provide a better understanding of the brain structure-cognition relationships and help shed light on the current taxonomy of attention.


Neural probe technologies have already had a significant positive effect on our understanding of the brain by revealing the functioning of networks of biological neurons. Probes are implanted in different areas of the brain to record and/or stimulate specific sites in the brain. Neural probes are currently used in many clinical settings for diagnosis of brain diseases such as seizers, epilepsy, migraine, Alzheimer's, and dementia. We find these devices assisting paralyzed patients by allowing them to operate computers or robots using their neural activity. In recent years, probe technologies were assisted by rapid advancements in microfabrication and microelectronic technologies and thus are enabling highly functional and robust neural probes which are opening new and exciting avenues in neural sciences and brain machine interfaces. With a wide variety of probes that have been designed, fabricated, and tested to date, this review aims to provide an overview of the advances and recent progress in the microfabrication techniques of neural probes. In addition, we aim to highlight the challenges faced in developing and implementing ultra-long multi-site recording probes that are needed to monitor neural activity from deeper regions in the brain. Finally, we review techniques that can improve the biocompatibility of the neural probes to minimize the immune response and encourage neural growth around the electrodes for long term implantation studies. In the To date, a wide range of neural electrodes have been used in basic neuroscience and neural prosthetic research (brain machine interfaces) starting with the early electrolyte-filled micropipettes , 34-37 aA number of reviews have been previously published for neural probes and neuroMEMS by Najafi , Banks , Heidusc2.Neural probes are microstructures that form the connection between the biological neural tissue with physical devices and electronics. Implantable neural probes for neuroscience and brain machine interfaces are generally preferred to have a minimum footprint as possible to minimize neural damage and incorporate structural features that facilitate easy entry and movement through the brain tissue. The smaller footprint for the neural probe will allow the probe to approach the target neurons as closely as possible and thus improving the signal-to-noise ratio of the recorded activity or target a very specific region to be stimulated. Further, there is growing need to insert large numbers of accurately spaced recording sites into small volumes which is promoting the application of thin-film and micromachining techniques to the microelectrode production.Typically, a MEMS-based neural probe consists of a single or multiple long protruding structures or shafts (of lengths ranging from 200 μm to 15 mm) preferably with thin thickness (thicknesses ranging from 10 μm to 200 μm). Each shaft includes the recording sites, interconnect traces and back carrier area carrying the bonding pads to connect the probes to electronics. Ideally, shafts with minimum thickness are preferred to reduce damage to the tissue when the probe is inserted into the brain. Moreover, miniaturization of the neural probes is very crucial to approach and efficiently interface the neural tissue at the scales of neural cells whose size typically range between 10 and 50 μm in diameter and spaced 20 nm apart . In contMany research groups investigating neural probe technologies are faced with different challenges including non-standard and unconventional fabrication processes leading to low yield and high cost, lack of on-site and monolithic Integrated Circuit (IC) integration leading to high noise and reduced sensitivity, shorter sized probes mainly limited by fabrication technology, low design flexibility, and limited selection of materials having the mechanical properties that fulfill both the implantation application requirements and being compatible with standard microfabrication processing. In addition, biocompatibility of the neural probes to minimize the foreign-body immune response plays an important role in determining long-term function of neural probes during implantation. These parameters in combination ultimately determine the microfabrication process needed to manufacture the neural probes.3.Metal microelectrodes, which enabled the initiation of many pioneering studies in neural sciences, represent the most widely used neural probes . The eleet al. [Typically used electrode materials include platinum, iridium, platinum-iridium, gold, stainless steel, tungsten, and molybdenum. Quartz glass, Teflon, polyimide, and Parylene materials are used for the insulation of these electrodes. The insulating materials ideally should have a high dielectric constant in order to minimize stray capacitances affecting the electrode during recording. A multiple-electrode array of these insulated wires can be made by gluing individual metal wire electrodes together or by using cutoff wire bundles. For instance, Vassiliy Tsytsarev et al.  have devThe exposed or non-insulated area of metal wire electrodes which forms the recording site is further processed for manipulating the impedance of the site. For instance, Skrzypek and Keller covered a tungsten rod with polymethyl methacrylate (PMMA) and placed it in a Scanning Electron Microscope (SEM) such that the desired area on the probe tip was exposed by manual control of the SEM electron beam. The sample probes are shown in et al. has reported a floating array of metal probe by employing laser machining of a ceramic substrate to form recording holes [et al. as shown in More recently, Musallam ng holes . The lasAlthough metal based electrodes are still employed successfully in both acute and chronic recording from the brain tissue, these kinds of electrodes can record activity only at their exposed tip. Increasing the number of recording sites requires increasing the number of electrodes which results in a linear increase in overall probe size and causes undesirable neural tissue damage. Further, it is difficult to insert a large number of probes accurately into a small volume of brain tissue. The main advantage of metal electrodes is the simplicity in their manufacturing. This simplicity also leads to its disadvantage which is the lack of common standards and automation in electrode manufacturing from one institution to other. Due to the lack of automation, the electrical characteristics of the recording tip vary from one microelectrode to the next within a batch, from batch to batch, and from one laboratory to other.et al. [The micromachining of metal wire based neural probe arrays has been demonstrated using electroplating technique as it provides high aspect ratio structures for various metals. This process consists of fabricating multiple metal shafts on the same silicon substrate and then releasing them by back etching of the silicon. The electrodes with an insulation layer, consisting of Parylene-C as shown in et al.  employedet al. , 68. Theet al. .The fabrication starts with patterning multiple regions of seed layer with all except one layer being electrically isolated from the electroplating. The electroplated nickel which was used in the process will grow upward and outward from the edges of the patterned seed layer and results in a structure with rounded edges both in- and out- of plane as shown in During electrodeposition as the height of the electroplated metal equals the height of the neighboring electrically isolated region of the seed layer, plating will occur even on the insulated regions. The electroplating technique is mainly limited by anisotropic deposition rate due to the current crowding effect. Specifically, the electric field lines leave the cathode uniformly and crowd on the exposed metal regions on the wafer.The result is a higher deposition rate laterally than vertically. Moreover, wire-bonding cannot be used to connect the probes to external circuitry so wires have to be manually soldered to the probe bond pads. The silicon based probes discussed in the next section appear to be more attractive option than metal probes as each can carry many recording sites that are placed at relatively known positions on the probe. Thus, the increase in the number of the recording sites does not increase the overall probe size.4.The introduction of surface and bulk micromachining techniques played a significant role in standardizing the production of microprobes with very well defined shanks and precise placement of recording sites , 71. Thepia arachnoid membrane covering the brain. Silicon has well-recognized mechanical advantages suitable for neural probes and it allows the use of the well-established microfabrication technologies and equipment infrastructure developed for the integrated circuits industry leading to high precision and well-defined structural features. The use of silicon photolithography process allow excellent control over the recording site size, shape and spacing enabling multiple recording sites to be placed at variable heights on a single electrode shank. Such ability allows insertion of a large number of recording sites in a small volume which is not possible with metal wire arrays or bundles. Inclusion of integrated circuits can be performed directly on the probes and offers benefits in terms of better signal acquisition, reducing power line interference, and ultimately reducing the overall probe size by reducing the electrical cable sizes [The substrate forms an important part of the probe structure providing the mechanical support. Ideally, the substrate must be biocompatible, small enough to obviate damage to the tissue and strong enough to penetrate the le sizes -83.A well-known example of silicon based neural probes are the Michigan probes  and an epia layer of the brain needed special guide tools for insertion. Mechanical weakness of the probes causes the probes to crack and shatter and may cause severe damage and disturbance to the brain tissue during insertion. Another well-known silicon based-probes incorporating multiple-electrode arrays is the Utah probe and an example of which is shown in Michigan probes have been successfully used in a number of neuroscience applications, but they also suffer from some disadvantages related to probe thickness and durability. Typically, wet etching can be employed for limited probe thickness and thus we find that the typical thickness of the Michigan probes is 15 μm. These probes when pierced through the The Utah electrode arrays have a unique structure with the arrays pointing upwards  as opposed to all other silicon neural probes which are built lying down . As a result, the probe length of the Utah electrode arrays is limited by the silicon wafer thickness. The longest reported probe length is only 1.5 mm. Further, only one recording site can be made on each probe shaft and the fabrication process is not a typical batch process and therefore suffers from low production rates. The interconnection of the Utah electrode arrays is made of a set of polyimide insulated gold wires and ultrasonically bonded on the back of the array to a set of aluminum read-out pads. The stiffness of the metal wire bundle makes these unsuitable for chronic implantation in human brain and especially for the high-density electrode arrays.4.1.2) or glass sandwiched between a thin layer of silicon (device layer) and the silicon substrate (handle wafer). SOI based neural probes employ the SiO2 layer as an etch-stop layer. Several etching techniques are available where the etch rates for SiO2 are much lower than for silicon. The handle wafer is back-etched to release the probe shaft which means that the probe shaft thickness is defined by the thickness of the device layer. Different techniques have been used to develop neural probes using SOI wafers, starting with the use of backside wet-etching using potassium hydroxide (KOH) [SOI wafers are produced by placing a thin, insulating layer such as silicon oxide (SiOde (KOH)  solutionde (KOH) , 102.et al. [et al. and these fabricated probes are shown in et al. used double-sided DRIE of a SOI substrate with the buried SiO2 layer acting as an etch stop to pattern forklike probe shafts as shown in A combination of plasma etching (to shape the probe shafts) with KOH wet etching of the handle wafer (to release the shafts) was used by Kewley et al. . Anotheret al. . An impret al. used DRIE in combination with direct write laser lithography (DWL) for neural probe fabrication [More recently, Kindlundh rication . DWL is 4.2.There are many emerging MEMS and CMOS-MEMS processes that can be used to fabricate neural probes with specific features such as ultra-long reinforced structures and integrated signal processing capability. Typically, we find micromachined electrode arrays are constructed using silicon as the structural support layer with gold as the recording sites material. These materials are available in the standard MEMS processes such as SOIMUMPS from MEMSCAP and MicraGEM from Micralyne. Further, the use of standard fabrication processes yields mass-producible and well-defined probe structures. More recently, CMOS-MEMS process from DALSA 0.8 μm process with bulk micromachining can be employed for development of active and intelligent neural electrodes. However, post-fabrication processing is required for the CMOS-MEMS process to deposit gold or iridium on the electrode recording sites.The authors recently developed prototype neural electrodes using the MicraGEM process. The prototype includes high-aspect ratio silicon electrodes which have 10 μm thickness and up to 6.5 mm in length, although longer probes are possible. Ultra-long probes having lengths longer than 5 mm can be used for recording in deeper regions of the brain. Many probes developed previously for this purpose by several research groups failed (cracked or shattered) during implantation as they were unable to withstand the insertion axial forces, retraction forces, and tension forces of the brain tissue. To overcome the problem, the developed prototype investigates a novel principle of reinforcing the probes in their more susceptible areas to improve their strength during implantation without increasing the dimensions of the probe.Numerical studies are performed to determine the reliability of the ultra-long neural probes when subjected to significant shear stresses due to imperfect insertion into the brain and/or due to movement of the brain relative to the skull. The simulations are used to predict the behaviour of the probes when subjected to a buckling force under axial compression. The horizontal component of the force is very important for neural electrodes as it determines the force at which the probe tip penetrates the brain tissue. As seen from the simulation results in 5.The use of polymer materials for neural probes has been widely studied to replace the normal silicon nitride or silicon dioxide insulation layer deposited during the fabrication of silicon neural electrodes , 106-108et al. [et al. [et al. [et al. [Rousche et al.  demonstr [et al.  as shown [et al.  and is s [et al. .The flexible polyimide electrodes suffer from disadvantages due to the lack of rigidity which causes buckling during insertion into the brain. Given this reason, long length polyimide probes cannot be used as they bend due to lack of rigidity and reduces the accuracy in targeting a selected region in the brain. Moreover, polyimide-based probes are prone to failure due to possible moisture absorption by polyimide. Given this, silicon-based neural probes appear to be more successful owing to the mechanical properties of the silicon and the flexibility of patterning using standard microfabrication technologies.6.Perhaps the greatest challenge facing the use of neural implants is the resultant tissue response to the neural injury which effects the long term functioning of the implanted neural probe. Signal deterioration soon after implantation of arrays has been reported by many groups. For these devices to be useful, they should ideally remain functional indefinitely for enabling self-contained and self-governing brain machine interfaces. Signal deterioration has been attributed to the general immune activation of the brain in response to the presence of a foreign device. Insertion of the electrode ruptures the vasculature and destroys all neurons in its path. Immediately following the insertion of an array, an acute immune response is activated which facilitates the recruitment of glial cells . Activatet al. [Using immunohistology, Biran et al.  showed tet al. . The braet al.  and headet al. . This moet al. . Local det al. . Anti-inet al. . This meet al. recently showed that using nanostructured porous silicon (PSi) surface for implants improved biocompatibility [In contrast to directly battling the immune response, some groups have proposed using neural growth factors to encourage neural growth around the electrode. Kennedy used a glass cone filled with growth factor releasing tissue and showed that using neural growth factors (NGF) improves recording over many months . In additibility . They retibility , 126-1287.The paper aimed to give an overview of the commonly used microfabrication technologies to develop neural probes for prosthesis and brain machine interfaces. Technological advancements in the areas of microelectronics along with surface and bulk micromachining have led to the development of a great number of passive and active probes. In the review, we described metal wire, silicon, and polymer based neural electrodes. Silicon based probes appear to be the most versatile and widely accepted technology for neural microelectrodes. We notice that there are three main considerations for the development of neural electrodes, namely, (i) probe material selection, (ii) microfabrication process, and (iii) biocompatibility interface. The combination of these three factors ultimately determines the functionality, usability, and long-term implantation reliability of the neural electrodes.Implantable neural probes are generally preferred to have a minimum footprint as possible to minimize neural damage and to facilitate easy entry and movement through the brain tissue. The above requirement combined with the growing interest to undertake neuroscience studies from deeper regions of the brain has necessitated the need to develop ultra-long probes (lengths longer than 5mm) with thicknesses less than 50μm. Such high aspect ratio structures pose a great design challenge as the probes have to be able to withstand the insertion axial forces, retraction forces, and tension forces of the brain tissue. New techniques such as probe reinforcement as described in this review have to be developed for these emerging applications. In near future, the standardization of commercial MEMS processes and emerging CMOS-MEMS processes could lead to the development of new neural probes that are cost-effective and mass-produced with ultra-long probe shafts and on-site/on-probe signal processing circuitry.