Prolonged exposure to stress has a wide effect on the brain and cognition. Error processing, as one of the crucial components of executive function, plays an important role in cognitive and behavior control. However, to date, there is little research addressing the relationship between chronic perceived stress and error processing. The present study aims to explore the relationship between chronic perceived stress by the Cohen Perceived Stress Scale (PSS) and different stages of error processing by the method of Event-Related Potential (ERP). The error processing was tested in a classical auditory Go/NoGo paradigm, and ERP components including early Error-related Negativity (ERN) and late Error Positivity (Pe) were computed as the indices of error processing. The results showed that the PSS score was positively correlated with the Pe amplitude but not with the ERN amplitude. The correlation between PSS and the Pe amplitude holds true even after controlling the trait anxiety and depression symptoms. These results suggest that the higher the chronic stress level, the more sensitive the individuals are to their own errors as well as the more emotional/motivated attention the individuals distributed to their own errors. However, longstanding animal and human studies have shown that prolonged exposure to stress may have a detrimental influence on mental and physical health4. In healthy individuals, chronic stress impairs prefrontal-dependent executive function such as working memory5, attentional control6 and decision making7, while enhances amygdala-dependent emotional processing such as exaggerated emotional response8, emotional learning9 and aggressive behavior10.Inevitably, we will encounter various stressful events in our daily life. Acute and moderate stress may cause adaptive stress response and allocate limited cognitive resources to the brain regions and organs that are currently demanded, thus increasing vigilance in response to changes and threats in the environment14. Error is often considered a potential threat and may activate defensive motivation systems to protect the organism. Detection of errors can improve future performance in general, while it has a survival significance in the environment full of threat and stress16.The ability to monitor one’s own behavior is another crucial executive function implemented by medial prefrontal cortex (mPFC) and anterior cingulate cortex (ACC), and it includes multiple dynamic stages such as continuous examination of ongoing behavior, augmented attention and alertness after mistake commission and mobilization of cognitive control to reduce the probability of committing more errors18. Following the ERN, the Pe is a positive ERP waveform, emerging at the centroparietal region between 200 to 400 ms after the onset of erroneous response. Studies indicate that the Pe reflects the conscious recognition of the error19 or the motivational/emotional evaluation18.The dynamic stages of error processing can be examined by event-related potentials (ERPs), and two specific response-locked components are the error-related negativity (ERN or Ne) and the error positivity (Pe). The ERN is a negative ERP waveform, appearing at the frontocentral region between 0 and 100 ms after the onset of the error commission, and the magnitude of ERN might reflect the automatic detection of error or the mismatch between actual response and required response22; while individuals with externalized behaviors like antisocial behavior, substance abuse and aggressive personality had impaired behavior monitoring and a smaller ERN amplitude than healthy individuals24. Therefore, some researchers suggested the ERN as an endophenotype for psychopathology26. Similarly, studies with healthy individuals revealed that the magnitude of ERN amplitude is positively related to trait anxiety27, trait negative affect28 and trait behavioral inhibition29, and negatively related to impulsivity30. Unlike the ERN’s proneness to trait characteristic, the Pe seems to be influenced a lot by the individual’s state. The Pe was amplified when participants were over concerned about errors33. Moser and colleagues (2005) found a smaller Pe amplitude but equal ERN under threat condition (with the presence of a Chilean rose-haired tarantula) compared to the safe condition34. Another study with major academic examination as chronic stressor again found that exam group had a larger Pe amplitude but similar ERN than non-exam group35.Several clinical studies have found abnormal error processing at both stages in various psychiatric disorders. Patients with internalized disorders such as compulsive-obsessive disorder, anxiety and depression were sensitive to errors, revealing as a larger ERN amplitude than healthy controls36, socioeconomic stress37 or reporting negative life events38. However, it has been proposed that it might be that “perceived stress”, the general perception about how individuals find environmental demands exceed their capacity regardless of the type of the environmental demand, is linked with long-term stress response and physical disease40. Orem et al. (2007) found that the higher level of self-chronic perceived stress is associated with worse (longer reaction time) set-shifting performance41. Furthermore, on the brain structural level, a longitudinal study revealed that chronic stress measured by Perceived Stress Scale (PSS) over an approximate 20-year period predicted decreased grey matter volume in the prefrontal cortex and hippocampus, even after controlling several potentially confounding factors42. Functionally, Treadway and his colleagues (2013) also found that higher perceived stress by PSS was associated with blunted activation of medial prefrontal cortex following behavioral feedback43. In line with these results, it is possible that perceived stress is also associated with disrupted prefrontal-dependent error processing.Measurements of chronic stress on human in recent studies have been heterogeneous and included different definitions such as academic examination44, a widely-used instrument to measure the perceived predictability, controllability and overload of daily life over the past one month period. The error processing was evaluated by the classical Go/NoGo task. The Go/NoGo task is a well-established measure for both correct and failed inhibitory control across neuroimaging and ERP studies46. Given the evidence reviewed above that chronic stress is associated with diminished brain function on behavioral, structural and functional levels, we hypothesized that chronic perceived stress is related with abnormal error processing.Therefore, to test the possibility, the aim of the present study was to investigate the continuous spectrum of chronic perceived stress levels, as opposed to categorized groups, in order to explore its relationship with error processing at neural level by ERP method. To address this question, chronic perceived stress was assessed by the PSS (10 item version)The mean PSS10 score was 16.08 (SD: 5.36). The mean CES-D score was 11.52 (SD: 8.317). The mean trait anxiety score was 42.68 (SD: 7.36).The mean omission error rate to Go trials was 8.86% (SD: 8.95), and participants responded with an average RT of 403.58 ms (SD: 66.39 ms). Commission errors occurred on 25.17% of NoGo stimuli with an average of 30.2 (SD: 19.48) false alarms to NoGo trials per participant.p-values showed that the ERN amplitude to erroneous response is larger than to correct response (−9.607 μV (SD: 5.20) vs. 0.499 μV (SD: 2.108), F = 217.504, p < 0.001). The mean latency of the ERN was 48.14 ms (SD: 33.40). The topographic distribution for the peak ERN . The topographic distribution for the Pe . No significant associations were found between CES-D or trait anxiety score and the Pe amplitude . PSS10, CES-D or trait anxiety scores were not associated with the ERN amplitude and latency . PSS10 was marginally positively correlated with false alarm rate . CES-D or trait anxiety scores were not associated with the hit rate and RT .There was a significantly positive relationship between PSS10 and the Pe amplitude  = 2.619, p = 0.06) above-and-beyond all the following step 1 covariates. Only PSS10 significantly predicted the Pe amplitude , in which they showed that the Pe amplitude was larger while the ERN did not differ compared the exam group who went through a long-term preparation for an important academic examination to the control group35. However, many factors like self-perceived overall competence and motivation47 may differ between the exam group and control group. Furthermore, the authors also found that the self-reported importance of the academic examination was significantly positively correlated with the Pe amplitude35. Therefore, it cannot be ruled out that the increased Pe amplitude in the exam group they found is due to the difference in academic motivation between the two groups rather than the chronic stress level. The present study, however, by using the perceived stress scale excluded the response variability within human individuals to the same stressor, for example, preparing for an academic examination. Humans appraise the uncontrollability, unpredictability and overload of their life by higher-order neocortical processing, which is proposed to activate the hypothalamic-pituitary-adrenal axis and stress response49. Moreover, by using continuous variables rather than categorical method improved statistical power and reduced parameter estimation bias50. In addition, the positive associations between chronic perceived stress and Pe amplitude were replicated after controlling depression and trait anxiety within prediction models. These results provided direct neurophysiological evidence for the association between perceived stress and error processing in a non-clinical sample of young males.Chronic perceived stress positively predicted the late stage of error processing rather than the early stage, i.e., the higher the level of chronic perceived stress, the larger the Pe amplitude. This result was consistent with the result from Wu 18. The increase in Pe amplitude with higher chronic perceived stress may indicate that the higher of chronic perceived stress, the more an individual is aware of the errors they made and in turn, the more emotional/motivational attentional resource they will allocate to the errors. Since error is considered as potential threat16, this interpretation echoed with the proposal that individuals under chronic stress are more sensitive to negative stimuli51. High perceived stress has been found linked to physical health like a higher risk of cardiovascular disease, hypertension as well as shorter telomere length52. Wirkner and colleagues (2019) found that higher chronic stress level measured by hair cortisol level was associated with enhanced emotional arousal toward affective stimuli as indicated by late positive potential53. Furthermore, imaging studies have consistently revealed that brain regions that process attention, executive control and emotion are influenced by chronic perceived stress. For example, Lederbogen and colleagues (2011) found that compared to the non-urban citizens, urban citizens who had higher chronic stress level showed larger amygdala response when confronted with acute stress54. Higher self-reported chronic stress was associated with lower activation of the ACC as well as the lower connectivity between amygdala and ACC during odor perception55. On the structural level, higher self-reported chronic stress was associated with smaller volume in mPFC and ACC in a cross-sectional study56, and even predicted decreased volume in orbitofrontal cortex in a longitudinal study42. Considering the general regulatory role of ACC in behavioral monitoring and emotion57, the positive correlation between chronic perceived stress and the Pe amplitude in the current study might be due to the chronic stress-induced excessive activation of the amygdala with reduced ability of the ACC to regulate the overactivated amygdala, leading to exaggerated emotional assessment of the errors. Interestingly, the level of chronic perceived stress was related with the Pe amplitude rather than the ERN component, suggesting chronic stress has a distinct influence on different stages of error processing. Previous studies have suggested that the ERN may reflect stable factors. For example, the ERN amplitude was positively correlated with trait anxiey27, trait negative affect28 and behavioral inhibition29 and negatively associated with impulsivity30. The result of the genetic study from Fallgatter et al. (2004) to some extent supported this hypothesis, in which they found that the ERN but not Pe amplitude was associated with the short allele of the serotonin transporter. Correspondingly, several studies found that the Pe component is more inclined to be affected by environmental factors and psychological states35. The current study provides more evidence that chronic stress is associated with the late stage  rather than the early state  of error processing. Future research might be better able to address this distinction by twin studies.According to the previous literature, the Pe might reflect conscious recognition of errors and the level of subjective motivation and emotional assessment of errors58. On the other hand, oversensitive to mistakes might also constitute a risk factor for mental illness such as mood and anxiety disorders25. Epidemiological studies showed that individuals who perceive negative experiences to be uncontrollable and overwhelming are more likely to develop depression after stressful traumatic events60. The link between higher perceived stress and psychiatric symptoms might partially be mediated by oversensitivity to the errors they made in daily life. Therefore, our results highlight the potential therapeutic value for stress-reduction interventions by adjusting individual’s error evaluation process.Our results may have some implications. The relationship between chronic perceived stress and error monitoring suggests that individuals perceived more chronic stress might be more sensitive to negative events such as committing an error. This increased sensitivity to errors, on the one hand, can promote behavioral adjustment to avoid making more errors in the future61, and it was proposed that it is the stress appraisal and coping causes long-term strain and finally lead to psychological and physiological disease62.There are some limitations need to be addressed in the current study. First, the sample of this study was male university students. Therefore, the results we obtained here might not be able to generalize to other groups. Secondly, the chronic stress level was assessed by a subjective perceived stress scale without physiological indicators such as hair cortisol. However, it is worthwhile to mention that people might appraise the impact and severity of the similar negative life events differentlyIn summary, the present study help identify how variation in perceived chronic stress influence neural response involved in error processing. Understanding the relationship between perceived chronic stress load and error processing in healthy people without the contamination of psychiatric-related factors is important to understand stress-related psychopathology. Our findings revealed that chronic perceived stress was associated with the later stage rather than the early stage of error processing, suggesting that the higher level of chronic perceived stress, the more an individual is aware of the errors they made and in turn, the more emotional/motivational attentional resource they will allocate to the errors.63, only male students were recruited through advertisements in a local university. Prescreening excluded people with acute or chronic psychiatric, physiological or neurological disorders. Sixty male students with normal auditory ability participated in the experiment. All participants were medication free and without smoking or drinking habit. The average age of the participants was 21.15 years (standard deviation (SD): 0.83, range: 20~23 years) and the average education year was 13.55 .This study is part of a larger project addressing the relationship between cognitive function and stress response. Due to the well-documented sex differences in stress response46 while EEG was recorded. For the auditory Go/NoGo task, the targets (Go stimuli) were 1000 Hz pure tones and the non-targets (NoGo stimuli) were 1032 Hz pure tones . The sound pressure was adjusted to a comfortable level. All the tones were presented binaurally over headphones. After an initial practice block of 10 trials, two experimental blocks each including 310 trails (20% NoGo and 80% Go probability) were completed with 2 min breaks between blocks. The duration of the tones was 200 ms and the stimulus-onset asynchrony randomized from 900 to 1100 ms. During each trial, one of the two tones were presented. Participants were instructed to respond as fast as possible by pressing a button on the keyboard once they heard the Go stimuli. The sequence of Go and NoGo stimuli was pseudorandom thus the consecutive presentation of two NoGo stimuli was avoided.At arrival, participants took a rest and filled out questionnaires on the computer. Subsequently, participants were seated on a comfortable chair in a normally illuminated room to perform a classical auditory Go/NoGo task44. This standardized instrument evaluates how much an individual perceives life as unpredictable, uncontrollable and overloading, which are the core components of the stressful experience and physiological stress response49. The scale measures perceived stress over the last one month on a 4-point Likert scale ranging from 0 (never) to 4 .Chronic perceived stress was assessed by the 10-item PSS (PSS10)64. Respondents indicated the extent of the symptoms they had experienced in the past week on a 4-point scale .Depressive symptoms were assessed by the Center for Epidemiologic Studies Depression Scale (CES-D)65.Trait anxiety was also measured by the trait subscale from the State-Trait Anxiety Inventory (STAI)22.CES-D and trait anxiety were used as covariates in our data analysis to correct for their potential relationship with perceived stress and error processingThe EEG was recorded with Ag/AgCl electrodes from 64 scalp sites mounted in an elastic cap  according to the international 10–20 system, with online reference to the left mastoid. Data were re-referenced offline to the average of both mastoids. One pair of electrodes placed above and below the left eye was used to record vertical eye movement and another pair placed 10 mm from the outer canthi of each eye to monitor horizontal eye movement. The impedance from all electrodes was below 5 KΩ. Signals were amplified with bandpass filter at 0.05–100 Hz and digitized at 1000 Hz.66, and time-locked to the onset of button press.The EEG data was processed by Scan 4.3 software . The data was further filtered with a 30 Hz lowpass filter. Ocular artifacts were removed from the EEG signal by a regression procedure implemented in the Neuroscan software. Trials with artifacts were automatically rejected with a criterion of ±100 μV. EEG data were epoched into periods of 1000 ms, including 400–200 ms before response as baseline according to previous studiesFor the behavior data, reaction time (RT) of the correct response to the Go trials was measured relative to stimulus onset. There were two types of accuracy evaluated: omission errors which calculated as the percentage that the participants did not respond to the Go trials and commission errors  which defined as the percentage of erroneous responses to the NoGo trials. Trials with RT below 50 ms and above 1000 ms were excluded.46, the peak amplitude and latency of the ERN at FCz and Cz sites where the ERN has its peak was detected 50 ms before and 150 ms after a false alarm response, and the mean amplitude of the Pe at CPz and Pz sites was measured between 300 and 500 ms after a false alarm response. To reduce statistical error, the average ERN from FCz and Cz and the average Pe from CPz and Pz were calculated to conduct the following correlational analysis.For the EEG data, only error responses of the NoGo trials  were averaged for the ERN and Pe. According to our own data see Fig.  and prev2 (ΔR2) in step 2. All the statistics were conducted by SPSS23.0, and two-tailed tests with the threshold for significance was set to p < 0.05.Due to the non-normal distribution of the ERP data, nonparametric Spearman correlations were performed to assess relationships between ERP indices of error processing  and scores of psychological scales. In subsequent 2-step hierarchical regression analyses, we tested whether chronic perceived stress predicted the error processing independently of additional confounding factors. For these 2-step regressions, the error processing index which had a significant relationship with psychological measurements was entered as dependent variable. In step 1, we entered trait anxiety and depressive symptoms; in step 2, we entered the PSS10 (our indicator of chronic perceived stress). The unique percentage of variance of error processing explained by chronic perceived stress was calculated by the change in RAll participants gave written informed consent and were paid for their participation. This experiment was approved by the Ethics Committee of Human Experimentation in the medical department at Shenzhen University. The experiment was conducted in accordance with relevant guidelines and regulation.


SHANK genes code for synaptic scaffolding proteins at excitatory synapses and mutations in all SHANK genes have been associated with autism. Here, we present three behavioral aspects of the mutant mice deleted for exon 16 in Shank2. First, we treated Shank2 mutant mice with methylphenidate to rescue the hyperactivity. Our failure to do so suggests that the hyperactivity displayed by Shank2 mutant mice is not related to the one displayed by the typical mouse models of hyperactivity, and might be more closely related to manic-like behaviors. Second, by testing the effect of group housing and social isolation on social interest, we highlighted that Shank2 mutant mice lack the typical flexibility to modulate social interest, in comparison with wild-type littermates. Finally, we established a new protocol to test for social recognition in a social context. We used this protocol to show that Shank2 mutant mice were able to discriminate familiar and unknown conspecifics in free interactions. Altogether, these studies shed some light on specific aspects of the behavioral defects displayed by the Shank2 mouse model. Such information could be used to orient therapeutic strategies and to design more specific tests to characterize the complex behavior of mouse models of autism.Mouse models of autism can be used to study evolutionarily conserved mechanisms underlying behavioral abnormalities in social communication and repetitive behaviors.  SHANK family is of interest since mutations in each of the three members of this family  have been identified in patients with autism, but with a gradient of severity  are characterized by atypical social communication, including social interactions and verbal and non-verbal communication, as well as stereotyped and repeated behaviors and restricted interests . More thseverity . PatientIn the present study, we focused our experiments on mice lacking the Shank2 protein. To date, three different genetic constructions of the model exist , a treatment for individuals diagnosed with attention-deficit/hyperactivity disorder (ADHD)  highlighted that the habituation-dishabituation protocol was ethologically relevant. We adapted this protocol by testing simultaneously the mutant mouse and the wild-type mouse to control rigorously the investigation of social recognition in Shank2 mutant mice.In this study, we aimed at modulating the behavioral phenotype of the r (ADHD) . To testShank2+/+, 10 Shank2+/-, 10 Shank2-/-; treatment: 10 Shank2+/+, 10 Shank2+/-, and 10 Shank2-/-) and females  aged of 4–6 months. Female mice were housed in groups of 2–4 per cage, while males were single-housed because of high aggressiveness. Shank2 mutant mice were initially described in Shank2+/- and Shank2-/- mice treated with either saline solution or methylphenidate using non-parametric Wilcoxon-Mann-Whitney U-tests given the small sample size and the non-normal distribution of the data.We tested males  since we cannot distinguish the identity of the caller in such a setting. Nevertheless, previous studies suggested that the contribution of the new comer is minor in comparison with that of the occupant  or call duration was longer than 5 ms and the frequency range was larger than 6.25 kHz and there was only one direction of frequency modulation (upward or downward);- “complex”: frequency modulations in more than one direction and frequency range larger than 6.25 kHz (modulated), or inclusion of one or more additional frequency components  but no constraint on frequency range (complex);- “unstructured”: no pure tone component, “noisy” calls;- “frequency jumps” (fq jumps): presence of one or more jump(s) in frequency, without time gap between the consecutive elements; it can include noisy parts within the pure tone call or not.We used unpaired non-parametric Wilcoxon tests to compare the latency for the first contact, the total time spent in contact, the latency for the first call and the call rate between genotypes within the social condition (either grouped or isolated). We used paired non-parametric Wilcoxon tests to compare the latency for the first contact, the total time spent in contact, the latency for the first call and the call rate between social conditions (grouped or isolated) within genotypes. We used Chi-squared (with a Bonferroni correction for multiple testing) tests to compare the proportions of calls within and between social conditions or genotypes.Supplementary Material – Review of social recognition protocols). We adapted the habituation-dishabituation protocol by testing a pair of female mice (one Shank2+/+ mouse and one Shank2-/- mouse) with a juvenile C57Bl/6J female mouse as the stimulus mouse. By testing paired mice, we collected data from the mutant and its control mouse in exactly the same conditions. We tested 12 pairs of one Shank2+/+ adult female and one Shank2-/- adult female (6 months of age), paired together at least 1 week before testing. Juvenile females were housed in groups of four upon arrival at the animal facility (3 weeks of age) and until the end of the experiment. Mice were identified through RFID chips implanted under isoflurane anesthesia and after local sub-cutaneous injection of lidocaine (at 4 weeks of age for the juveniles and at weaning for the tested mice). On the day of testing, mice were habituated to the testing room 20 min before the test. We then introduced the pair of Shank2 mice in a test cage . The pair of mice was left to explore freely the test cage for 20 min. After this habituation time, we introduced an unknown C57Bl/6J juvenile female mouse (5 weeks of age) and let the mice freely interact for 2 min. After this first encounter, we took out of the cage the juvenile mouse and left it alone in a housing cage for 5 min. We then re-introduced it in the cage for 2 min. We repeated these steps for four encounters with the same juvenile. On the fifth encounter, we introduced another unknown C57Bl/6J juvenile female mouse (5 weeks of age) from another housing cage. During each encounter, a tracking system, Live Mouse Tracker, allowed to individually follow each of the three mice and to automatically extract the time spent in contact with the juvenile for each tested mouse  for each juvenile encounter. We used paired non-parametric Wilcoxon tests to compare the total time spent in contact within each genotype between the fourth encounter and the fifth one.We used paired non-parametric Wilcoxon tests to compare the total time spent in contact between All experiments involving animals complied with the European ethical regulation, and were validated by the ethical committee CETEA n°89, Institut Pasteur, Paris. These procedures were realized within the project APAFIS#7706-20 161 12317252460 v2.-Shank2/- mice are highly hyperactive in comparison with their wild-type littermates  and Shank2+/- mice . In addition, Shank2+/- mice also displayed hyperactivity but to a lower extend in comparison with their wild-type littermates . These results confirmed the initially described gene dosage-related hyperactivity in this model  and by Shank2+/- mice  in comparison with saline-injected mice of the same genotypes in both sexes. The MPH treatment also increased significantly the distance traveled by -Shank2/- female mice in comparison with saline-injected -Shank2/- female mice . The difference in -Shank2/- male mice in comparison with saline-injected -Shank2/- males was not significant , but the hyperactivity was already very high and we might have a saturation effect. In both sexes, MPH-injected -Shank2/- mice still traveled significantly longer distances in comparison with MPH-injected Shank2+/+ mice .termates . Here, wis model . MPH treShank2-/-, Shank2+/-, and Shank2+/+ mice reacted similarly to the injection of MPH, with an increase in their locomotor activity. MPH was therefore not efficient to rescue hyperactivity in this model.Overall, Shank1 mutant mice , in the time spent in contact , in the latency for the first call , and in the vocal repertoire used . The only difference was found in the number of vocalizations emitted, with less vocalizations emitted by -Shank2/- mice in comparison with wild-type mice .In this study, we therefore compared social interactions and ultrasonic vocalizations in the occupant-new comer test between group-housed and isolated adult female mice from the -Shank2/- mice spent significantly shorter time in contact with the new comer  and emitted less ultrasonic vocalizations in comparison with Shank2+/+ mice . Interestingly, when considering the ultrasonic vocalization repertoire used, -Shank2/- mice emitted significantly more calls from the short and unstructured categories than Shank2+/+ mice in the isolated condition . In contrast, the duration of the calls for each call type did not differ significantly between social conditions or between genotypes .When females were isolated for 3 days, Shank2-/- and Shank2+/+ mice only after social isolation, and not when animals were group-housed. These results suggested that -Shank2/- mice were impaired in their modulation of social motivation.Overall, significant differences in social interaction and communication emerged between Shank2-/- female mice displayed impairments in social recognition in the three-chambered test, with no significant preference for an unknown conspecific in comparison with a familiar conspecific  interacting repeatedly with a C57Bl/6J juvenile female. After four encounters with the same juvenile, the pair of mice encountered an unknown juvenile on the fifth encounter.In their initial characterization, specific . Howeverild-type . In the Shank2+/+ mice spent in contact with the juvenile decreased over the four successive expositions to the same juvenile . Then, they spent a significantly increased time in contact with the second juvenile presented for the fifth encounter , suggesting that they were able to discriminate their juvenile conspecifics. -Shank2/- mice followed a parallel profile despite the fact that they tended to spend shorter time in contact with the juveniles in comparison with their wild-type littermates . As in Shank2+/+ mice, the time that Shank2-/- mice spent in contact with the juvenile between the fourth and the fifth encounters also increased significantly .The social recognition protocol appeared to be valid. Indeed, the time that Shank2-/- mice spent shorter time in contact with the juvenile in comparison with wild-type mice but remained able to differentiate two individuals.Overall, the The psychostimulant methylphenidate inhibits the reuptake of dopamine and norepinephrine (increase of dopamine and norepinephrine extracellular levels) . It is k-Fmr1/- mice , while this hyperactivity was rescued by the mood stabilizers valproic acid or lithium , suggesting an atypical reward seeking behavior. In parallel, clioquinol, a zinc chelator and a ionophore mobilizing trans-synaptic zinc has been shown to rescue the reduced social interest in the three-chambered test in mouse models of autism, but could not rescue the hyperactivity observed in the Shank2Δex15-16 model  and also with isolated mice.Whether the behavior of the -Shank2/- mice. It shows that the hyperactivity displayed by this model cannot be reduced by methylphenidate. It also highlights that the social deficits displayed by this model might stem from a lack of flexibility in the social motivation. Finally, it provides more details on the social recognition deficits, which were not visible during direct social interactions with a conspecific, i.e., when the tested mice had access to all identity cues of the conspecifics. Interestingly, the reduced social interaction displayed by -Shank2/- mice might therefore be misinterpreted as a problem in social cognition. We propose an alternative hypothesis that -Shank2/- mice have high capacity to integrate social cues, but once these social cues are integrated, the mice display less social motivation to interact. Such information could be used to orient therapeutic strategies and to design more specific behavioral tests to characterize mouse models of autism.To summarize, the present study gathers new pieces of information on specific aspects of the behavior of the The raw data supporting the conclusion of the manuscript will be made available by the authors, without undue reservation, to any qualified researcher.EE, NT, A-TF, and JL-S performed and analyzed the experiments. A-MLS performed the genotyping of the mice. FdC created the tracking system for the social recognition experiments. TMB generated the mouse model. EE and TB conceived the project and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Contrast sensitivity (CS), the ability to detect small spatial changes of luminance, is a fundamental aspect of vision. However, while visual acuity is commonly measured in eye clinics, CS is often not assessed. At issue is that tests of CS are not highly standardized in the field and that, in many cases, optotypes used are not sensitive enough to measure graduations of performance and visual abilities within the normal range. Here, in order to develop more sensitive measures of CS, we examined how CS is affected by different combinations of glare and ambient lighting in young healthy participants. We found that low levels of glare have a relatively small impact on vision under both photopic and mesopic conditions, while higher levels had significantly greater consequences on CS under mesopic conditions. Importantly, we found that the amount of glare induced by a standard built-in system (69 lux) was insufficient to induce CS reduction, but increasing to 125 lux with a custom system did cause a significant reduction and shift of CS in healthy individuals. This research provides important data that can help guide the use of CS measures that yield more sensitivity to characterize visual processing abilities in a variety of populations with ecological validity for non-ideal viewing conditions such as night time driving. Vision represents our main modality of perception and interaction with the surrounding environment. While visual acuity (VA) is often considered the gold standard for vision assessment, contrast sensitivity (CS), defined as the ability to detect or discriminate low contrast gratings, may provide a more informative index of functional vision in both clinical and healthy populations . For exaTo obtain a broad-spectrum characterization of CS across multiple scales and frequencies, several clinical and laboratory tests have been developed to measure the CS function (CSF). While the CSF is generally measured in a low light environment, changing ambient lighting conditions can affect CS measurements in distinct and clinically relevant ways . RecentlThe CSF is impacted not just by changes in ambient lighting, but also by different sources of light that can interact with the optics of the eye to induce functional vision disability due to a phenomenon called glare. For example, upon leaving a dark room to go outdoors, one can experience a period of impaired vision due to the bright daylight, in what is called adaptation or discomfort glare. Another type of glare is the so-called disability glare, which is due to excessive exposure to light under dark conditions  or to the scatter of light rays due to increasing ocular opacity , which can impair functional vision and reduce sensitivity to contrast , posing in situ keratomileusis (LASIK) surgery . Using this device, In the present paper, we first measured the photopic CSF of a group of young healthy individuals with and without glare for the standard luminance level provided by the CSV-1000E (Experiment 1). In Experiment 2, we measured the mesopic CSF of another group of young healthy individuals with the CSV-1000E with and without glare. The rationale behind Experiment 2 was that mesopic condition resembles a number of possible real-life situations such as night driving, thus representing an ecologically informative scenario. The results from these first two comparisons showed that the luminance level offered by the CSV-1000E is not sufficient to induce a statistically significant reduction of the CSF in photopic or in mesopic viewing condition in young healthy participants. Participants in Experiment 2 were also tested under mesopic light condition at a higher luminance level (225 lux), obtained with a custom-built glare-inducing system, and results revealed that the CSF was significantly reduced at this elevated luminance level.Finally, as a follow up we tested a subgroup of participants from Experiment 2 on four additional intermediate luminance levels  and found that illumination levels of at least 125 lux were sufficient to induce significant glare and a reduction of mesopic CSF in healthy participants. Overall, these results suggest that under both photopic and mesopic conditions, the CSV-1000E cannot induce glare that significantly impairs CS in young healthy individuals. However, the use of higher luminance glare, obtained with our custom-built apparatus, was capable of significantly reducing CSF where 125 lux was found to be the sufficient level of intensity needed to reduce CSF in most individuals – a light intensity that is substantially higher than offered by current standard commercial tests.2. CSF measurement was conducted under photopic condition (luminance level 10 to 108cd/m2) for Experiment 1 and under mesopic (luminance level 0.001 to 3 cd/m2) in Experiment 2 and 3. Two glare conditions were tested in Experiment 1: no glare (standard CSF measurement) and glare . Experiment 2 utilized a custom built glare system that had four led light placed at the each corner of the device and angled to face to participant that was viewing the CS unit . Three glare conditions were tested in Experiment 2: no glare, low glare (69 lux LED light source), and high glare (228 lux LED light source). In Experiment 3, a subgroup of participants of Experiment 2 were tested on four additional glare levels , to better characterize the modulation of CSF induced by different levels of glare intensity. Mesopic vision was obtained with 1.5 neutral density filters.A VectorVision CSV-1000E  CS unit was used for the test. The CSV-1000E is a translucent retro-illuminated chart that consists of a series of circular achromatic sine-wave patches. The unit self-calibrates to produce a mean luminance of 85 cd/m53 participants  took part in Experiment 1, and 71  in Experiment 2. A subgroup of 10 participants from Experiment 2 took part in Experiment 3. All participants were recruited at Western University of Health Sciences, Pomona CA. The study was approved by the Institutional Review Board and all participants provided written informed consent to participate in the study. The study was conducted in accordance with the Declaration of Helsinki (1964). The data management was adhered with Health Insurance Portability and Accountability Act (HIPAA) regulations. All participants had normal or corrected-to-normal vision of 20/20 or better on ETDRS acuity chart. They took part in two testing session of about 30 min each.Participants were tested at a distance of 2.5 m from the CSV-1000E. This distance allowed measuring spatial frequencies of 3, 6, 12, and 18 cycles per degree (cpd). The CSV-1000E is composed of a series of circular achromatic sine-wave patches 1.5′ in diameter. Across each row, there are vertical pairs of circles, one of which contains the sine-wave patch while the other is blank but has the same luminance as the test patch. There are four rows, each corresponding to one of four spatial frequencies. When selected, a given spatial frequency is rear-illuminated and a suprathreshold example of the test pattern is shown to the participant. Each spatial frequency is presented at eight different contrast levels that systematically decrease from 0.045 to 2.00, 0.7 to 2.20, 0.78 to 2.26, 0.6 to 2.08 and 0.3 to 1.81, respectively, in eight columns from left to right. The participant is asked to indicate whether the given test pattern is located in the top or bottom patch. The contrast threshold is defined as the contrast of the last column the subject could correctly identify the location of the sine-wave patch. The average inter-stimulus drop in contrast is 0.15 log units between steps 2 and 8; the contrast change between step 1 and step 2 is 0.3 log units.F3,150 = 427.16, p<0.001, η = 0.895) but no effect of glare condition on CS , demonstrating there was no statistically significant difference between CS measurements obtained with and without glare under photopic conditions .For Experiment 1, we conducted a two-way repeated measures ANOVA with factors Glare condition (Glare vs. no glare) and Spatial Frequency . Results showed a main effect of Spatial Frequency , Spatial Frequency  and a significant interaction Glare condition × Spatial Frequency . Post hoc tests (Holm–Bonferroni-corrected t-tests) showed that the CSF in the high glare condition was significantly different (lower) from both no glare and low glare conditions (both p<0.001).For Experiment 2, we conducted a two-way repeated measure ANOVA with factors Glare condition  and Spatial Frequency . Results showed a main effect of Glare condition , while there was no difference between no glare and low glare condition for any of the SF .1,74 = 46.04, p<.0001, η = 0.384), the results showed a main effect of SF , meaning that glare affected differently the various SFs tested.In order to better understand the effect of glare on SF, we performed an additional repeated measure ANOVA on the difference between baseline and low glare (No glare score–Low glare score) and baseline and high glare (No glare score–High glare score). Alongside the main effect of Glare level , meaning that glare reduced CS significantly more at lower spatial frequency than higher .To further explore the effect of glare on SF, we ran Holm–Bonferroni corrected multiple comparisons between the SF levels for the No glare-High glare differences. This test showed that the difference between high glare and baseline for the lowest SF (3 cpd) was significantly larger than for the higher spatial frequencies  and Spatial Frequency . Post hoc tests (Holm–Bonferroni-corrected t-tests) showed that except for the lowest level of glare , all the other intensities induced a significant reduction of CS with respect to the no glare condition . While all light intensities greater than 69 lux induced a general reduction of CS measurements, the greatest reduction across all spatial frequencies was observed for the highest intensity level (225 lux).Finally, for Experiment 3, we conducted a two-way repeated measure ANOVA with glare condition  and Spatial Frequency  as factors. Results showed a main effect of glare condition  with a custom-built setup. In Experiment 1, we tested the CSV-1000E under glare condition, while in Experiment 2 and 3 we used the custom-built glare-inducing system that allowed for higher luminance levels compared to the CSV-1000E. Results showed that the level of glare induced by the latter (69 lux) was insufficient to affect the CSF under photopic (Experiment 1), and mesopic viewing conditions (Experiment 2) in young healthy participants. However, a higher level of luminance intensity (225 lux) under mesopic viewing condition reduced CS significantly at all the spatial frequencies tested (Experiment 2), and especially at lower spatial frequencies (3 cpd). A further experiment (Experiment 3), aimed at measuring a range of illumination intensities in mesopic light condition confirmed results obtained in Experiment 2 and showed that values above 125 lux did induce a significant decrease of the CSF. Taken together, the present results suggest that, while glare-induced reduction of the CSF can be achieved with currently available systems in clinical populations, i.e., astigmatism patients who underwent LASIK surgery , in ordeThe evidence that the relatively low intensity of luminance offered by the CSV-1000E induces glare in post-surgery patients  but not As stated in the introduction, reducing CS with glare presents a number of advantages: On one hand, it would allow to simulate a series of real life scenarios in which glare disrupts vision ; on the other hand, it would provide a more sensitive assessment tool for the visual evaluation of patients recovering from eye surgery. Additionally, reducing the baseline CSF would increase the dynamic range of the test and reduce the influence of ceiling effects to measure subtle changes in groups with an already high-level of vision . Current market offers VA and CS tests that, while integrating artificially inducing glare equipment, do not seem to be effective in a healthy population.Indeed, CS tests like the CSV-1000E offer a limited number of contrast levels per SF, therefore a young healthy observer or an athlete who wants to monitor his visual abilities might reach the lowest level of contrast provided by the test already during baseline measurements. Consistently, 3 out of 10 participants in Experiment 3 reached the lowest contrast value for 3 cpd already in the first measurement. A procedure that reduces the observer’s initial CSF would then provide a larger number of contrast values beyond his threshold, allowing to measure performance improvements beyond a level that without glare would be at ceiling.Participants in Experiment 2 and 3 were tested under mesopic vision. The rationale was that in both normal sighted individuals and patients, it seems to be the light condition under which the CSF is mostly affected , in partThis is due to the fact that under cone vision, gain control mechanisms maintain perceptual constancy for contrast over a wide range of illumination levels, avoiding response saturation . On the The practical applications of simulating glare under this lighting condition appear evident when one considers how both the rate  and the In conclusion, it is possible to significantly reduce the CSF of young healthy individuals using artificially induced glare; however, this requires a level of luminance that is about 2–3 times higher than what CS tests currently offer on the market today. Of note, we nonetheless showed that it is possible to induce a CSF reduction with an illumination intensity 10 times lower than what has been proven to be effective in a previous studies (125 lux vs. 2000 lux and 4000 lux; PGD designed and implemented the experiments, and data was collected by the technicians in his lab. MM analyzed the data. All authors interpreted the results, and wrote and reviewed the main manuscript.PGD has received funding support and is a consultant to VectorVision not related to present study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chrysophrys auratus (snapper) to 1) construct the first linkage map for this species, 2) scan for growth QTL, and 3) search for putative candidate genes in the surrounding QTL regions. The newly constructed linkage map contained ∼11K SNP markers and is one of the densest maps to date in the fish family Sparidae. Comparisons with genome scaffolds of the recently assembled snapper genome indicated that marker placement was mostly consistent between the scaffolds and linkage map (R = 0.7), but that at fine scales (< 5 cM) some precision limitations occurred. Of the 24 linkage groups, which likely reflect the 24 chromosomes of this species, three were found to contain QTL with genome-wide significance for growth-related traits. A scan of 13 candidate growth genes located the growth hormone, myogenin, and parvalbumin genes within 5.3, 9.6, and 25.0 cM of these QTL, respectively. The linkage map and QTL found in this study will advance the investigation of genome structure and aquaculture breeding efforts in this and related species.Characterizing the genetic variation underlying phenotypic traits is a central objective in biological research. This research has been hampered in the past by the limited genomic resources available for most non-model species. However, recent advances in sequencing technologies and related genotyping methods are rapidly changing this. Here we report the use of genome-wide SNP data from the ecologically and commercially important marine fish species  New QTLe.g., a linkage map or high quality genome assembly) is an important prerequisite for QTL mapping, as it allows the relative positioning of different marker loci. High-quality genome assemblies are most effective because they allow genetic markers to be positioned at a base-pair level, while also providing sequence information for the surrounding area. However, most non-model species do not currently have chromosome-level genome assemblies and instead rely on linkage maps to ascertain the relative position of markers in the genome  in a wide range of taxa , commonly referred to as the Australasian snapper (henceforth referred to as “snapper”). Snapper supports a valuable recreational and commercial inshore fishery around the northern parts of New Zealand, southern Australia, and some of the Pacific Islands  construct a high density linkage map, 2) conduct QTL mapping for three measures of growth , and 3) investigate the position of 13 candidate growth genes and their relative position to growth QTL.Here we focus on the marine teleost 1 = 70 individuals, F2 = 577 individuals) were investigated in this study. Uncontrolled mass spawning of the F1 generation in a single tank was used to produce the offspring F2 generation. This resulted in a complex pedigree, meaning that we obtained a combination of full-siblings, half-siblings, and unrelated individuals in the F2 generation (Supplementary Table 1). The F2 offspring were held in a single tank until they were approximately one year old and then split evenly among four tanks with comparable feeding, light, water flow, aeration, tank design. All research carried out in this study was reviewed and approved by the animal ethics committee of Victoria University of Wellington in New Zealand (Application number 2014R19).A snapper breeding program was started at The New Zealand Institute for Plant & Food Research Limited in 2016 and includes a population with three generations held at the Nelson Research Centre in New Zealand. Data from the two most recent generations  and again when they were approximately three years old (1045-1131 days). Length measurements were made by collecting images of each individual and then making measurements from those images. A ruler was included in each image to provide a scale. The number of individuals measured differed between year one and year three as a result of natural mortality during the study.Samples of fin tissue were collected for all fish and DNA was extracted from these samples using a modified salt extraction protocol . QuantifPstI and MspI. The adaptor ligation step was done after digestion, without allowing the DNA/adaptor mixture to dry out. The barcoded adaptors were designed by Deena Bioinformatics and bound to the PstI cut sites. Adaptors were subsequently annealed according to the method of Ko et al. (2003). The high fidelity enzyme AccuPrime Taq DNA polymerase High Fidelity (Life Technologies) was used for amplifications. Each library was amplified separately and its quality assessed by capillary electrophoresis prior to sequencing . All GBS libraries were prepared in parallel in plates. Duplicate or triplicate samples were prepared for each of the parent and grandparents and single samples for each of the offspring . Each plate  was pooled, then cleaned up, quantified and sent to the Australian Genome Research Facility (AGRF) in Melbourne, Australia, for sequencing. Each pool was sequenced on a single lane with the Illumina HiSeq 2500 platform in single end (SE) mode, with a read length of 100 bases. In total, eight pools of libraries were sequenced in eight lanes for this project.Only high quality genomic DNA was used for the preparation of Genotyping By Sequencing (GBS) libraries based on the protocol described by FastQC was used to conduct an initial check of the sequencing data quality. Sequences were then de-multiplexed and cleaned. Adapters and primers were removed and the sequencing data were cleaned using Fastq-mcf in the ea-utils package . Genotyp2 individual in the dataset were identified using CERVUS v3.0.7  were used, and included a total of 269 offspring and 14 parents, and reduced the total number of available SNPs for this analyses to 20,311 SNPs. Markers were separated into chromosomes with the SeparateChromosomes module (logarithm of odds (LOD) limit = 14, minimum markers per linkage group = 50). The marker order was then generated with the OrderMarkers module. Markers near the start and end of each linkage group (start and end 10% based on centimorgan (cM) distance) were removed if they were more than 3 cM from the next closest marker. The accuracy of the final linkage map was investigated by comparing the linkage map position (cM) with the position of markers on available genome scaffolds (base-pairs) from the genome assembly (number of scaffolds 5998). The scaffold and base-pair position for each marker in the linkage map was retrieved from the STACKS v1.40 output files. Using this information the correlation between linkage map (cM) and scaffold (base-pair) position was tested for all scaffolds that contained >50 SNPs. The mean and 95% confidence interval of the correlation residuals was then calculated. Whether scaffolds were placed uniquely on one of the 24 expected linkage groups was also investigated as well as the number and total base-pairs of scaffolds able to be positioned on the linkage group. The extent of linkage disequilibrium across the linkage groups was reviewed by calculating the pairwise linkage disequilibrium results for each set of markers using PLINK v1.9 . The sex-specific recombination rate was calculated by comparing the linkage map distance (cM) and genome scaffold distance (bp) between individual marker pairs for males and females.The parents for each FINK v1.9 . This wa1 and F2 generations and phenotyping data from the F2 generation were used. QTDT used 10,716 markers which had been placed on the linkage map. GRIDQTL used a subset of markers (n = 1007), which were filtered randomly to a minimum spacing of 1 cM. Before running the analysis, the genotype data were filtered for Mendelian errors by dropping loci for any individual that contained alleles not observed in either of the two parents. The phenotype measurements used for the analysis were standardized by tank and date collected to correct for temporal and tank effects. The QTL scan results from QTDT were visualized using the ggplot2 library v3.1.0 in the R statistical environment v3.2.3  and using the built in permutation procedure in GRIDQTL with 1000 permutations.Quantitative trait loci identification was carried out using the general model implemented in QTDT v2.6.1 . Strong positive correlation was also observed between year one and year three for each measure .Peduncle length, fork length, and weight were recorded when individuals where 436-487 days old (year one) and 1045-1131 days old (year three). The distribution and relative sizes of fish in year one and year three are illustrated in 1) and 23.9x in the parents (F2).A total of 1.6 billion reads were produced for all eight pooled GBS libraries with approximately 2, 4 or 6 million reads for each single, duplicate, or triplicate individual library respectively. Using the STACKs pipeline a total of 20,311 SNPs were found after filtering for >7x coverage, present in 75% of the individuals in the population, and a minor allele frequency (MAF) of 0.05. The average coverage per SNP was 15.6x in the offspring  and 30.2 cM from the nearest QTL peak (GRIDQTL). Myogenin on linkage group 3 was 9.6 cM from the nearest QTL marker (QTDT) and 18.4 cM from the nearest QTL peak (GRIDQTL). Paravalbumin on linkage group 16 was 25 cM from the nearest QTL marker (QTDT) and 7.8 cM from the nearest QTL peak (GRIDQTL).The base-pair position on the genome scaffolds were found for all 13 candidate genes including valbumin . Based oChrysophrys auratus. Proof checking the marker order against the snapper de novo genome assembly indicated that the linkage groups were of high quality. QTL mapping revealed eight markers on three linkage groups that were significantly associated with growth. Three candidate genes for growth were located on the same linkage groups as these QTL. These genomic resources will be used to inform the selective breeding program in New Zealand and will form the basis of further genomic investigation in snapper.We assembled the first chromosome level linkage map for the Australian snapper Salmo salar) includes ∼96K markers, although it should be noted that the genome size in this salmonid species is also significantly larger (2.97 Gb genome size)  . ComparaGasterosteus aculeatus, 3.11 cM/Mb, . It is not known how sex is being determined in snapper, but if the heterochiasmatic sex commonly has a lower recombination rate, then this may indicate that males are heterogametic in this species. Further work is needed to explore this in more detail.Using the newly constructed linkage map and available genome scaffolds, we were able to calculate the sex-specific recombination rates for snapper, which showed that females have a higher recombination rate compared to males . This reflects observations in other fish species, with females often . Other factors that can affect growth include feed amounts, fish density in tanks, and tank design . We attempted to control for these factors in the current study by standardizing the conditions between tanks and by standardizing measures from each tank.The target trait for this study was growth , which wSalmo salar: 0.06 to 0.08, , brill to 0.23, .e.g., candidate genes or causative alleles) are likely to be located. If high rates of linkage are present between markers, a confidence interval for the QTL region can be estimated - as seen in R/QTL , growth hormone inhibiting hormone (GHIH), growth hormone (GH), and insulin-like growth factors (IGF-1 and –II) (growth hormone and insulin-like growth factor I and II were able to be mapped to the linkage map in the current study. Growth hormone was located near (within 5.3 cM) a QTL of genome-wide significance. In Sparus aurata, a close relative of C. auratus, a microsatellite repeat in the promoter region has previously been implicated for differences in growth  are another set of potential candidate genes  was located on the same linkage group as a genome wide significant QTL, but was much further away from a putative growth QTL (25 cM) than the previous two genes. A mutation in the promoter region of this gene was previously found to be involved in weight differences in the finfish species Lates calcarifer.Previous studies have outlined a range of genes and molecular networks that are thought to be candidates for further investigation in teleost species . We locaand –II) . Of theste genes . These rWhile the linkage map constructed in this study can confidently place SNPs in ∼5 cM regions, further work is needed to improve the accuracy of marker placement. More accurate placement of SNPS would help with future work to fine-map and further characterize the QTL and candidate gene locations described in this study. Improved precision should be possible in the near future using the genome assembly that is being further improved by our group. Future work should also aim to detect possible sex-linked markers, to identify regions associated with sex determination, and to investigate sex-specific recombination patterns across the genome. While sex-specific information was not investigated in the current study, this is an area of particular interest in snapper and the data from this study could be used to further investigate it. In conclusion, this study provides valuable genetic and genomic resources for future evolutionary studies and aquaculture breeding programs in this and related species.


Restoration of sinus rhythm in atrial fibrillation (AF) by radiofrequency catheter ablation (RFCA) is associated with a transient stunning of left atrial (LA) function. However, the long-term effects of different ablation strategies on LA function remain undetermined. We performed randomized controlled trial to evaluate the effects of RFCA, cryoablation, and 3D mapping-guided cryoablation on LA function of proximal AF patients within 1 year. The 3D mapping-guided cryoablation was defined as a maximum of two cryoablation procedures for each pulmonary vein accompanied by RFCA for additional points until complete pulmonary vein isolation was achieved. Conventional and speckle tracking echocardiographic analyses were performed to evaluate LA function. Among the 210 patients (70 in each group) included, a trend of decreasing LA systolic and diastolic function was observed in all groups, as evidenced by decreases in peak A-wave velocity, the global LA peak systolic strain, the peak strain rate, the peak early diastolic strain rate, and the peak late diastolic strain rate within 7 days to 3 months after ablation followed by gradual recovery thereafter. However, the temporal changes in the above four strain parameters among the three groups did not differ significantly within 1 year after ablation . Parameters of the LA emptying fraction and LA dimensions were not significantly affected. These results suggested that stunning of LA function occurred within 7 days to 3 months after ablation, and different strategies of AF ablation did not differentially affect the temporal changes in LA function up to 1 year after ablation. Patients with AF have a significantly increased risk of thromboembolism events and heart failure, which makes the disease an important cause of mortality worldwide2. Currently, pulmonary vein isolation (PVI) by means of catheter ablation has become an important treatment for patients with drug-refractory paroxysmal AF (PAF)3. Conventionally, PVI is achieved by radiofrequency catheter ablation (RFCA). It has been recognized that restoration of sinus rhythm in AF is associated with a transient stunning of left atrial (LA) function, which may be associated with an increased risk of thromboembolic events5. Our recent study in PAF patients undergoing RFCA indicated that dramatically reduced LA function occurred within 7 days after ablation and was accompanied by an increased prothrombotic tendency6. However, the long-term changes in LA function following RFCA remain unclear.Atrial fibrillation (AF) is a common atrial arrhythmia that is prevalent particularly in the elderly7. By single energy release for lesions encircling the pulmonary vein (PV)8, the efficacy of cryoballoon ablation for AF has been confirmed in previous studies9. However, for some AF patients (15%10 and 28%11 according to previous studies), unrecognized incomplete PVI may persist despite the application of a standardized protocol for cryoablation and the use of a circular mapping catheter to record the LA-PV potential connections. For these patients, 3D mapping-guided cryoablation complemented with RFCA of additional points may improve the efficacy of cryoablation. However, the long-term temporal changes in LA function of AF patients following cryoablation and cryoablation complemented with RFCA, to the best of our knowledge, have been rarely evaluated. The aim of the study was to evaluate the effects of cryoablation, 3D mapping-guided cryoablation, and RFCA on long-term atrial function in patients with PAF.Cryoballoon ablation has emerged as a novel ablation strategy in patients with PAF2) as compared with that in the Freeze 3D (69.0 ± 56.0 mGycm2) and RF groups . Moreover, perioperative complications such as phrenic nerve injury and vascular injuries were not significantly different among the three groups.Overall, 210 patients (mean age: 59.1 years) with PAF were included in the current study, with 70 randomized to each group. The baseline characteristics of the included patients are shown in Table The temporal changes in peak A-wave velocity did not differ significantly among the three groups . Therefore, we observed trends in temporal changes of the above parameters with a combination of the three ablation groups. We found that the εP, SRP, SRE and SRA at both the two-chamber and four-chamber views were significantly decreased at day 7 after the ablation procedures for included patients with the combination of the three ablation groups  and systematic inflammation (hs-CRP) within 4 weeks after PAF ablation are shown in Fig. In this study, we found that for PAF patients undergoing catheter ablation, a transient decline in LA function occurred within 7 days to 3 months after ablation, which is consistent with previous notions that LA function is stunned after the restoration of sinus rhythm in AF. Moreover, we found that the temporary changes in LA function as evaluated by conventional and sparkle tracking echocardiography were not significantly different among patients who received ablation via different strategies, including conventional RFCA, novel cryoablation, as well as 3D mapping-guided cryoablation complemented by RFCA for additional points. Interestingly, we also found that although cryoablation was associated with more complete myocardial damage as compared with RFCA, systematic inflammation was not significantly induced. Moreover, pilot results showed that the three ablation strategies did not show significant differences regarding the recurrence of ATA within 1 year or the incidence of severe complications during the perioperative period. Based on the above findings, we conclude that PAF ablation with RFCA, cryoablation, or 3D mapping-guided cryoablation did not differentially influence long-term atrial function after AF ablation. The value of 3D mapping-guided cryoablation for PAF remains uncertain at the current stage.14. We found a trend of transient decreases in peak A-wave velocity, global LA peak systolic strain, peak systolic strain rate, peak early diastolic strain rate, and peak late diastolic strain rate from 7 days to 3 months after ablation followed by gradual recovery of these parameters, indicating the occurrence of LA stunning in these patients during this period. These results confirm our previous findings that for PAF patients who receive RFCA, reduced LA function could be observed within 7 days after restoration of sinus rhythm6. Moreover, we expanded these findings by showing that LA stunning, characterized by reduced LA systolic and diastolic function, could be observed within 7 days after cryoablation or cryoablation guided by 3D mapping. These results may reflect the previous notion that LA stunning is a common outcome of sinus restoration rather than a feature of a certain ablation strategy. Interestingly, in view of the fact that LA stunning has been related to the risk of thromboembolism events5 and the relapse into AF15 after sinus restoration, the results of our study may indicate that the risk of thromboembolism events related to AF recurrence may not differ fundamentally in patients who are treated using the three different treatment strategies. This has been confirmed by the results of the Fire and Ice study9 and recent meta-analyses18. Taken together, these results demonstrate that the temporal changes in LA function after ablation are not significantly affected by the ablation strategies, highlighting the importance of adequate anticoagulation therapy and rhythm monitoring despite the immediate success of AF ablation in these patients.One of the important strengths of our study is the comprehensive evaluation of the temporal changes in LA function following PAF ablation in patients who received ablation using different strategies. Despite the use of the conventional echocardiographic measurements of LA function based on pulsed-wave Doppler techniques, we also applied speckle tracking analyses to dynamically evaluate the movements of the atrial myocardium. This technique has been recommended as a novel measurement method for LA function because it is angle-independent, real-time, and quantitatively reflective of the global and strain systolic and diastolic function of LA19. These findings may be reflective of the unique feature of cryothermal energy-induced histological changes of the myocardium. Changes in myocardial tissues in response to cryoablation, as compared with RFCA, include ice crystal formation and preserved tissue architecture, but more extensive ablation lesions to the myocardium20. However, the clinical relevance of more complete myocardial ablation but relieved systematic inflammation caused by cryoablation remains to be understood.Another interesting finding of our study is that although cryoablation was associated with more complete myocardial damage as compared with RFCA, systematic inflammation was not significantly induced. This finding is consistent with the results of a previous study in Europe, which also showed that cryoballoon ablation of AF causes more significant myocardial damage but does not induce a significantly greater inflammatory response as compared with RFCA11, we designed a 3D mapping-guided cryoablation complemented by RFCA for additional points. Although we did not find improvements in atrial function or ATA recurrence during follow-up in patients allocated to the 3D mapping-guided cryoablation group, the incidence of AF recurrence seemed to be lower in patients who received 3D mapping-guided cryoablation (9.5%) as compared with that among those who received cryoablation (16.0%) or RFCA (20.4%). Studies aiming to improve the success of cryoablation for AF by using mapping-guided ablation are rare. In a cohort study from the USA, using of CARTO or NavX mapping system as a guiding strategy to cryoballoon ablation yielded longer fluoroscopy times without an improvement in AF recurrence21. However, another pilot study using a high-resolution mapping system during cryoablation for AF significantly improved the detection of incomplete PVI, which is expected to reduce the incidence of unrecognized incomplete PVI-related recurrence of AF11. Overall, the optimal strategy for guided cryoablation and its potential value in clinical practice remain uncertain at present, and more clinical trials are warranted.In view of previous reports that considerable patients experience incomplete PVI despite the use of the circular mapping catheter to record the LA-PV potential connections during cryoablation for AFOur study has limitations that should be noticed when interpreting the results. Firstly, our study was designed to evaluate the effects of different ablation strategies on LA function rather than on clinical outcomes such as AF recurrence. Therefore, large-scale trials are needed to evaluate the influences of different ablation strategies on clinical outcomes such as thromboembolism events and AF recurrence in the future. Secondly, use of 3D mapping-guided cryoablation leads to additional expense. The most cost-effective ablation strategy for PAF patients remains to be determined. Thirdly, the study was a single blind clinical trial, which could lead to additional bias, especially considering it involved a small number of patients. Fourthly, the application of RFCA for patients in the Freeze 3D group may also have confounded the outcome of LA function in this study. Finally, this was a single-center study. The results of our study should be further confirmed by large-scale multi-center studies.In conclusion, the results of our study showed that LA stunning occurred within 7 days to 3 months after ablation regardless of the ablation strategy applied, and different strategies of AF ablation did not significantly affect the temporal changes in LA function for up to 1 year. Further studies are needed to determine the optimal strategy to guide cryoablation and its potential value in clinical practice.http://www.chictr.org.cn, No. 16007954, firstly registered on 20/02/2016). All the methods were conducted according to the CONSORT statement22.This study was designed to evaluate the potential influence of three different ablation strategies , applied with and without perioperative statins, on the long-term LA function in PAF patients. Therefore, six groups were allocated (three ablation arms with and without statins), and in this paper, we focused on the reporting of the influences of the ablation strategies on LA function. The protocol of this single blind, randomized controlled trial was approved by the Ethics Committee of the Second Hospital of Hebei Medical University, and we hereby confirm that all methods were performed according to the guidelines and regulations approved by the Ethics Committee. Informed consent from the included patient was required. Participation in the study was voluntary and the patient could withdraw their consent at any time. The study protocol was registered at Chinese Clinical Trial Registry  ECG-confirmed PAF that occurred at least twice within 6 months before study enrollment; (2) occurrence of PAF remained despite application of class I and III antiarrhythmic drugs; and (3) <80 years old and agreed to receive catheter ablation treatment for PAF. Patients with the following clinical statuses were excluded: (1) prior history of receiving catheter ablation for AF; (2) atrial thrombosis; (3) diagnosis of valvular heart disease ; (4) an LA dimension of >50 mm; (5) prior history of prosthetic heart valve replacement; (5) pregnancy; or (6) existing liver and kidney diseases, malignant tumors or hematological system diseases.Patients were randomized to one of the following groups according to computer generated random sequences: (1) the cryoablation group (Freeze group): AF ablation was achieved via a standardized protocol with the first or second generation cryoballoon; repeated cryoablation was performed for each pulmonary vein until complete PVI was achieved; (2) the cryoablation combined with 3D mapping group (Freeze 3D group): cryoablation was performed a maximum of two times for each pulmonary vein under guidance with a 3D mapping system with the first or the second generation cryoballoon; if PVI was not achieved, RFCA was applied for the additional points until complete PVI was achieved; and (3) the RFCA group (RF group): AF ablation was achieved via a standardized RFCA procedure. The clinical characteristics and routine biochemical indexes of each patient were obtained before the ablation procedures. Only the physicians who performed the ablation procedures were aware of the grouping of the patients.Reconstructive computed tomography (CT) images of the PV were obtained from all included patients before the performance of the ablation procedure.For patients allocated to the Freeze group, single-balloon ablation with a cryoballoon either 28 mm or 23 mm in diameter  was performed according to the results of angiographic images of the PV. Briefly, an inner lumen mapping catheter  was placed into each PV ostium. Then, cryoballoons of selected diameters were advanced and placed at each PV ostium. Cryothermal energy was released and the single-time cryoablation was performed for up to 240 s for procedures of the first-generation cryoballoon and up to 180 s for the procedures for the second-generation cryoballoon. The cryoablation could be repeatedly performed until complete isolation was achieved.For patients allocated to the Freeze 3D group, a circular mapping catheter  was used to construct the configuration and build the structures of the left atrium and PV guided by the EnSite NavX 3D mapping system. Then, cryoablation was performed as described in the Freeze group up to two times for each PV. If PVI was not completely achieved, RFCA was applied for the additional points until complete PVI was achieved.For patients allocated to the RF group, a standardized RFCA procedure was performed with a mapping catheter  and a 3D electro-anatomical mapping system . The procedures of ablation were performed by a single group of four physicians for all of the included patients according to the randomized group allocation, which was led by a Chief physician (Dr. Ruiqin Xie).After discharge, the patients were followed up in the clinic at 1, 2, 3, and 4 weeks and 2, 3, 6, 9, and 12 months after the procedure. Patients were followed up for at least 1 year after the ablation for PAF. The primary outcome of the study was the change in LA function as evaluated by real-time echocardiographic examination. Moreover, we evaluated the influence of different ablation strategies on markers of myocardial injury  and systematic inflammation  within 1 month after the procedures. In addition, the influence of different treatment strategies on the recurrence of atrial tachyarrhythmia (ATA) during follow-up was also explored in PAF patients in each group. Recurrent arrhythmia was defined as any ATA lasting for at least 30 s.Echocardiographic examination was performed at baseline, at 1, 2, 3, and 4 weeks, and at 2, 3, 6, 9 and 12 months after PAF ablation using an echocardiographic system . The echocardiographic examination was performed by one experienced physician who was not aware of the grouping of the patients. Briefly, both the early (E) and late (A) diastolic filling velocities were obtained using pulsed-wave Doppler at the mitral valve leaflet tips. In the apical four-chamber view, the maximal LA volume was measured at the end of the left ventricle (LV) systolic phase just before the opening of the mitral valve, while the minimal LA volume was measured at the end of the LV diastolic phase just after the closure of the mitral valve. Subsequently, the LA emptying fraction (LAEF) was calculated according to the following equation  × 100. The LA maximum anteroposterior diameter was measured in the parasternal long-axis view, while the transverse and vertical diameters of the left atrium were measured in the apical four-chamber view.P), the peak early diastolic strain rate (SRE), and the peak late diastolic strain rate (SRA) were measured during LV ejection, early diastole, and late diastole (occurring after the P wave), which indicate the LA reservoir, LA conduit phase, and LA active contraction phase, respectively.Subsequently, we analyzed the longitudinal LA strain using speckle tracking echocardiography . Briefly, strain data of the typical 12 LA segments  were averaged to estimate the global LA peak systolic strain (εP) during LV ejection (LA reservoir phase). The peak strain rate , and categorical variables are expressed as counts and percentages. Comparisons of continuous variables among the three groups were performed with analysis of variance (ANOVA), and for comparisons of categorical variables, chi-square analyses were applied. Mixed-effects models were constructed when comparing the effect of ablation strategy and time period. The ablation strategy was set to be a fixed effect, and the time period was set to be a random effect as the data were not collected at equal time intervals. For post-hoc analyses, Bonferroni and Dunnet adjustments were used as appropriate. A p value < 0.05 was considered statistically significant. SPSS software  was used for statistical analyses.


Although peripheral nerve stimulation using intraneural electrodes has been shown to be an effective and reliable solution to restore sensory feedback after hand loss, there have been no reports on the characterization of multi-channel stimulation. A deeper understanding of how the simultaneous stimulation of multiple electrode channels affects the evoked sensations should help in improving the definition of encoding strategies for bidirectional prostheses. We characterized the sensations evoked by simultaneous stimulation of median and ulnar nerves (multi-channel configuration) in four transradial amputees who had been implanted with four TIMEs (Transverse Intrafascicular Multichannel Electrodes). The results were compared with the characterization of single-channel stimulation. The sensations were characterized in terms of location, extent, type, and intensity. Combining two or more single-channel configurations caused a linear combination of the sensation locations and types perceived with such single-channel stimulations. Interestingly, this was also true when two active sites from the same nerve were stimulated. When stimulating in multi-channel configuration, the charge needed from each electrode channel to evoke a sensation was significantly lower than the one needed in single-channel configuration (sensory facilitation). This result was also supported by electroencephalography (EEG) recordings during nerve stimulation. Somatosensory potentials evoked by multi-channel stimulation confirmed that sensations in the amputated hand were perceived by the subjects and that a perceptual sensory facilitation occurred. Our results should help the future development of more efficient bidirectional prostheses by providing guidelines for the development of more complex stimulation approaches to effectively restore multiple sensations at the same time. Using different sensory encoding and stimulation strategies, information from sensors embedded into hand prostheses has been translated into sensations perceived by the users16. Through direct neural stimulation using peripheral neural interfaces, various types of sensations have been elicited, such as pressure, touch, vibration, paraesthesia, tingling, or electricity17.Major effort has been devoted to restoring sensory feedback in upper limb amputees18. This approach allows sensory feedback to be used in simulated daily living activities18 by exploiting multiple sensations through stimulating the median and/ or ulnar nerves18. This leads to an improvement in motor control19, as well as the ability to recognize the shape and compliance of objects15 or to simultaneously integrate position and tactile information21.Recent studies have shown that these elicited sensations can be used for the real-time control of hand prosthesesIn these studies, the sensations evoked through electrical stimulation were characterized by stimulating single active sites (ASs). Further two ASs (one from the median and one from the ulnar nerve) were combined in a multichannel setup. However, to the best of our knowledge no detailed characterization of the stimulation parameters and the sensations evoked for combined-channel configurations has been carried out so far. A deeper understanding of this mechanism could potentially increase the effectiveness of sensory feedback in sensorized prosthetic systems.22) . Two were implanted in the median nerve and two in the ulnar nerve , the pulse amplitude was modulated between 10 µA and 980 µA, whereas the pulse-width was fixed to between 20 µs and 120 µs. The frequency was fixed to 50 Hz. The interval between each pulse train was 2 seconds. The subjects were asked to report the location, extent, type, and strength of the sensation. The strength of the sensation was rated with a scale from 0 to 10, where 0 was “no sensation”, 1 was the sensation threshold, 10 was pain.Then, to assess the outcome of DCC and TCC, the ASs that were the most repeatable during SCC  in terms of reported sensation properties, were selected for combination. DCC stimulation took place with one AS stimulating in the median, and one AS in the ulnar nerve, or both ASs in one nerve. For TCC, two ASs of the median nerve and one AS for the ulnar nerve were used Table . The pulThe elicited sensation locations for SCC and DCC for all four subjects are shown in Fig. The results for subject 2 Fig.  show thaThe minimum charge to elicit a sensation for every subject with SCC and DCC stimulations is shown in Fig. The perceived sensation types related to all four subjects for SCC and DCC are shown in Fig. When stimulating in TCC (Subjects 1 and 4), the sensation location and type, as in DCC, were the result of the linear combination of the sensations evoked in SCC  was tested as the control condition for the median nerve stimulation.Figure In order to characterize multi-channel stimulation configurations for restoring sensory feedback in prosthetic users, four subjects were implanted with TIMEs in their median and ulnar nerves. We characterized these configurations both through subjective patient reports, and objective EEG measures.In all the subjects, the sensation type remained the same in SCC, DCC and TCC, and the location of the sensation obtained in multi-channel configurations turned out to be the combination of the sensation location elicited in single-channel configurations.23. As expected, by stimulating with two electrodes implanted in two different nerves, the fiber types addressed and numbers did not differ with respect to SCC. Interestingly, even when stimulating two electrodes that were placed in the same nerve (Subject 2), results were similar to when DCC involved two nerves.In fact, locations and types of sensations depend on the number and types of sensory fibers elicited by the neural stimulation24. However, this effect was anecdotally reported only by Subjects 1 and 2. We hypothesize that these reports were sporadic because during the characterization process, the sensations elicited were not always of exactly the same intensity25. This further shows that the TIMEs in combination with the neurostimulators create clear sensations with low crosstalk between channels26. Indeed, we excluded that the perception of a single central sensation relates to the summing of voltage fields from multiple channels resulting in activation of different neural populations than in single channel configurations, since the stimulation channels used were at the distance higher than 1.6 mm27 (apart for one TCC combination).It has been reported that when sensations from multiple points of the phantom are evoked at a very similar intensity, the subject has the illusion of perceiving only one sensation, which is the combination of the multiple points28. For the use of two or more channels together in a closed-loop neuroprosthesis, the right perceptual threshold must be set in order to correctly exploit the multiple sensory feedback during daily life activities.The thresholds of the injected charge reveal that DCCs caused a significant reduction in the threshold charge compared to the same channels used with SCCs. This result would seem to indicate a perceptual facilitation due to the simultaneous stimulation of two residual nerves. It has been proposed that the somatosensory cortex is not just a simple relay, but can actively contribute to the integration of somatosensory inputs30. This finding is in line with Forss et al.31, Maguiere et al.32 and Allison et al.33 in terms of median nerve stimulation, and reflects the event cascade that follows the processing of a somatosensory input.The EEG analysis showed that the tactile sensations generated by SCC, DCC and TCC stimulation elicited early SEP components. They are characterized by positive and negative deflections respectively on the contralateral parietal  and frontal  EEG derivations symmetrically distributed across the CP1- C4 line  needed for TCC to elicit a perceivable sensation might also have been a contributing factor for the observed reduced SEP amplitude.The TCC SEPs exhibit a smaller amplitude, which further suggests the presence of an early integration process similar to that of DCC. In addition, the amplitude distribution over the scalp channels of N20 for TCC Fig.  is very 17.Taking into consideration the decrease in thresholds when using SCC, with respect to DCC and TCC, a characterization of all three stimulation configurations should be performed to adapt the injected charges to different types of hand grasps . Moreover, complex algorithms using multi-channel configurations that can stimulate more than three ASs at a time could be developed to achieve more efficient stimulation strategies37.Our study revealed that single AS thresholds change with the configuration type  and so the characterization of the sensation has to be repeated for each configuration. To avoid this, an interleaved stimulation delivered through the different channels could be appliedFinally, to verify the effectiveness of sensory feedback systems that provide stimulation through more than three electrode channels, further experiments are required.Four transradial amputees were recruited for the clinical trials. Subject 1 was a 36-year-old male, who had undergone a left transradial amputation around 10 years before the experiments. Subject 2 was a 37-year-old male with a two-year old traumatic (car accident) transradial amputation  two years before the clinical trials. Subject 3 was a 53-year-old female amputee with a transradial amputation at the proximal third of her left arm, caused by an industrial accident. Subject 4, a 48-year-old female with traumatic transradial amputation of the distal third of her left forearm (dominant) occurred 23 years before the trials.Ethical approval was granted by the Institutional Ethics Committees of the Policlinic Agostino Gemelli at the Catholic University of Rome, Italy. The protocols were approved by the Italian Ministry of Health. Informed consent was signed by all patients. Throughout the entire duration of our study, all experiments were conducted in accordance with relevant guidelines and regulations. Informed consent was given for the publication of identifying information/images.7. In brief, a general anaesthesia was performed, during which an approximately 15 cm-long incision on the left arm was performed. After exposing the median and ulnar nerves, a microscope was used to implant one proximal and one distal TIME in each nerve  was connected to the TIME electrodes.To perform a characterization of the sensations evoked through electrical stimulation, a neurostimulator  or multi-channel (DCC and TCC) stimulation configurations were applied  and increased the stimulation amplitude in order to find the perceptual thresholds (from 10 µA to 980 uA). Then, if the perceptual thresholds were not found, we repeated the procedure with a higher pulse width. The perceptual threshold was the average of the charges at which the minimum sensation was reported by the subject. Location and extent were indicated on a picture of a hand, shown by the graphical user interface of our software. The type of the sensation was described by the subjects along with the other parameters and recorded through the software. At the end of the sensation characterization procedure, we found all the charges necessary to reach the perceptual thresholds for each active site. To assess the outcome of DCC and TCC, the ASs that were the most repeatable ones in the SCC characterization procedure were selected to be combined and used. Most repeatable ASs are the ones that do not or only slightly change during the characterization process in terms of reported sensation location, type and perceptual threshold. Trains of stimulation with increasing amplitude were delivered simultaneously from two (DCC) or three (TCC) active sites. The charge range identified during SCC characterization was used for each channel. Then, the threshold of the channels in DCC and TCC was the value of charge at which all the channels themselves elicited a sensation of minimum intensity at the same time. To determine such values, the minimum amplitude of stimulation of all the channels was slightly adjusted. As in SCC the single values of stimulation were delivered at least 3 times. For example: M2 AS5 (SCC) caused a sensation on the index finger. U1 AS12 caused a sensation on the little finger. When combining the two DCC, using the same thresholds as in SCC, the little finger was not perceived. By increasing the minimum delivered amplitude of current of U1 AS12 both sensations could be felt simultaneously. The sensations elicited by DCC and TCC were characterized with the same strategy as in SCC, i.e. by assessing the sensation location, type and perceptual thresholds. The tests took place for a one-month period for Subject 1, and five months for Subjects 2–4 who were implanted in different time periods. The four subjects were implanted over a time course of five years (2013–17). The following stimulation configurations were included: for subject 1 five SCCs and four DCCs were tested. With Subjects 2 and 3 six SCCs and four DCCs were tested, and with Subject 4 six SCCs and five DCCs were tested. TCC was tested with Subjects 1 and 4 only (2 active site combinations each). Table The interval between each pulse train was two seconds. After every stimulus, the subjects were asked to report the sensation location, extent, type and intensity. A custom-made software application was used to control the stimulator and to record stimulation parameters and subjects’ reports. When the amplitude was modulated, the perceived sensation intensity varied accordinglyNeural correlates of mono and multi-channel stimulation were investigated by acquiring 64-channel electroencephalographic (EEG) data. Subject 1 underwent one rest session (5 minutes) and several stimulation sessions, SCC-median, SCC-ulnar, DCC, and TCC lasting around 10 minutes each. During the recordings, the subject sat on a comfortable armchair and was instructed to maintain his facial muscles relaxed and avoid sudden head movements as much as possible.In the SCC-median condition, a 20nC charge was injected through AS M2 AS5 with reference to the GND of the same electrode. For the SCC-ulnar, 24nC were injected in U1 AS12. For the DCC, U1 AS12 24nC were applied, whereas M2 AS5 a charge of 17.5nC was injected (different GND for U1 and M2). For the TCC we used M2 AS5 a 17.5nC, U1 AS12 24nC and M2 AS14 with an injected charge of 12nC. M2 AS5 and M2 AS14 were referred to the same GND (median electrode). U1 used the GND of the ulnar electrode.40 was used, and electrode impedance was below 10kΩ in at least 95% of recordings throughout the whole experiment.The inter-stimulus interval was set to 850 ms. Signals were recorded using a 64 channel EEG amplifier (Brain Products BrainAmp) with a 1000 Hz sampling rate. A montage in accordance with the 5% 10/20 system41. Continuous data was processed using a reliable independent component analysis (RELICA) approach42 to remove artifacts and other non-neural noise sources, without any preliminary data43 dimensionality reduction. To maximize the stability and dipolarity44 of independent components (ICs), raw data was high-pass filtered using a zero-phase 1.2 Hz, 24th order Chebyshev type II filter, low-pass filtered using a zero-phase 45 Hz, 70th order Chebyshev type II filter and resampled at 250 Hz. Channels were removed that had a kurtosis outside five standard deviations with respect to other channels or with prominent prolonged artifacts as confirmed by visual inspection. Epochs with high-amplitude artifacts or high- frequency muscle noise were also identified by visual inspection and removed. The remaining data was submitted to RELICA with AMICA core45, and 100 point-by-point Infomax independent component analysis (ICA)46 bootstrap repetitions.The data was analyzed using custom Matlab  scripts based on routines from the EEGLAB toolbox47. Dipolar and stable ICs related to stereotyped artifacts such as movement and blinks were then removed from the data. Epochs from −100 ms to 300 ms with respect to the onset of each stimulation pulse were extracted and centered with respect to the pre-stimulus baseline. Similarly to continuous data, noisy epochs were rejected by careful visual inspection. The remaining trials for each condition were averaged to yield somatosensory event related potentials (SEPs). Scalp topographies were drawn by color-coding SEP values for each channel location at a particular latency based on amplitude .ICA unmixing weights were then re-applied to the raw data, this time high-passed filtered using a 0.5 Hz 94th order Chebyshev type II filter and a custom (complex poles only) 50 Hz comb notch filter48 adapted from the FieldTrip toolbox49.All sensation characterization data was extracted and processed in Matlab . The normality of the data was first checked (one-sample Kolmogorov-Smirnov test). Since none of the data was normally distributed, a Kruskal-Wallis test was adopted (instead of ANOVA). When necessary, a Tukey-Kramer correction for multi-group comparison was applied. α was set to 0.05. In order to compare the occurrences of sensation types, a Fisher’s test was run. α was set to 0.05. Regarding EEG analysis, significance of SEP differences across the stimulation types  for selected channels was assessed using Montecarlo statistics with multiple comparisons cluster correctionSupplementary material