Our assessments of effort are critically shaped by experiences of exertion. However, it is unclear how the nervous system transforms physical exertion into assessments of effort. Availability of the neuromodulator dopamine influences features of motor performance and effort-based decision-making. To test dopamine’s role in the translation of effortful exertion into assessments of effort, we had participants with Parkinson’s disease, in dopamine depleted (OFF dopaminergic medication) and elevated (ON dopaminergic medication) states, exert levels of physical exertion and retrospectively assess how much effort they exerted. In a dopamine-depleted state, participants exhibited increased exertion variability and over-reported their levels of exertion, compared to the dopamine-supplemented state. Increased exertion variability was associated with less accurate effort assessment and dopamine had a protective influence on this effect, reducing the extent to which exertion variability corrupted assessments of effort. Our findings provide an account of dopamine’s role in the translation of features of motor performance into judgments of effort, and a potential therapeutic target for the increased sense of effort observed across a range of neurologic and psychiatric conditions. However, increased dopaminergic availability has also been associated with increased effortful exertion and low-level features of motor performance6. These seemingly incongruous findings in the domains of effort-based decision-making and motor performance raise the question of how dopaminergic signaling might influence the translation of physical exertion into judgments of effort7.After a bout of physical activity, we assess how much effort we exerted and use this judgment to guide our decisions about future exertions. Previous work has shown that dopamine influences decisions between effortful exertion and reward by modulating reward value, without contributing to the estimation of effort cost8. It has been suggested that this dopaminergic facilitation of effort-based decision-making is the result of dopamine alleviating motivational deficits by increasing reward sensitivity, rather than decreasing effort cost sensitivity9. These results point to dopamine’s influence on reward signaling that is dissociable from effort signaling.Studies of the influence of dopaminergic availability on effort-based decision-making have shown that increased dopamine makes individuals more willing to exert effort for reward11. Relatedly, individuals with Parkinson’s disease (PD) exhibit a generalized slowness of movement  that is exacerbated when they are withdrawn from dopaminergic medication. It has been proposed that dopaminergic availability does not influence the organization of movement per se, but rather the underlying motivation to initiate movement10. Despite dopamine’s influence on motor performance, it is unclear how such dopamine-mediated effects might impact judgments of physical effort and decisions to exert.In the context of motor performance, increased dopaminergic availability has been associated with increases in physical effort exertion and the initial velocity of exertion13. During effort assessments, such exertion variability may increase uncertainty in performance outcomes, and disrupt accurate effort assessment. Our hypothesized role of dopamine is based on studies that have found relationships between low-level features of motor performance and dopaminergic availability11. It has been suggested that dopaminergic modulation influences the signal-to-noise ratio in the nervous system, contributing to enhanced motor performance and sensory acuity14. With this in mind, we reasoned that dopamine could modulate variability in effort exertion , which may lead to individuals’ more accurate assessments of effort.Here we investigated the influence of dopamine on the processes responsible for assessing effort levels following physical exertion. We hypothesized that variability in participants’ exertion would disrupt their assessments of effort, and dopamine would decrease both exertion variability and the extent to which variability inflates effort assessments. This hypothesis is motivated by studies showing that increased variability in motor output is associated with increased effort costsTo test these hypotheses, we evaluated the influence of dopamine availability on assessments of effort in persons with PD. Participants performed a series of assessments of their physical exertion under conditions of decreased  and increased  dopamine availability. The OFF and ON conditions were assessed on separate days. In each condition, participants were first trained to associate the grip force exerted on a hand-clench dynamometer with numeric effort levels between 0 and 100 Fig. . These e15, were included in the analysis for this study  and those in the OFF and ON conditions . This finding is consistent with previous studies of isometric force production which have shown that variability in motor output increases proportionally with the amount of exertion 18. In the PD group, we found that dopamine availability interacted with the effect of mean exertion on exertion variability . When participants were in the ON dopamine condition, they exhibited less of an increase in exertion variability as exertion increased, compared to the OFF condition. We found that control participants’ exertion variability matched participants in the OFF condition . These results align with our hypothesis of increased dopamine availability facilitating a reduction in exertion variability during effortful motor output.During the effort assessment phase, in both the OFF and ON dopamine conditions, participants were able to exert effort at the target levels Fig. . A trialt = 21.452, df = 2634, p = 3.1948e−94) PD participants in the ON and OFF conditions , recalled assessments of effort increased with their mean exertion. In the PD group, there was a significant interaction between dopamine treatment condition and mean exertion on assessments of effort , indicative of increased dopamine availability having a dampening effect on increases in effort assessment with exertion. To further assess how dopaminergic availability was related to participants’ accuracy of effort assessment we examined the slope of the relationship between participants’ mean exertion and levels of assessment - perfect effort assessment would result in a slope of unity. In the dopamine OFF condition, the average slope was significantly greater than unity , corresponding to overassessment of effort in a dopamine depleted state. In the dopamine ON condition, we failed to find a significant difference in slope from unity , suggesting that dopamine had a protective effect on effort assessment accuracy. Control participants’ effort assessments matched participants in the dopamine OFF condition , and assessments were lower in the dopamine ON condition compared to controls . This comparison of the Control and PD groups show that these behavioral effects are generally related to dopaminergic availability/depletion, and not specifically related to dopamine interacting with PD disease symptomatology. Overall, these results suggest with participants translate their motor output into assessments of effort more accurately when in a state of increased dopamine availability.During the assessment phase, we found that Control and . This normalized metric allows us to account for the expected increase in variability with exerted effort Fig.  and evalt = 3.804, df = 2634, p = 1.456e−04) and PD groups . During trials in which participants exhibited increased exertion variability, they had a more pronounced disagreement between their actual exerted effort and their assessed effort. This is consistent with the idea that increased exertion variability disrupted participants’ assessments of effort. Additionally, dopamine had a protective effect on the propensity for exertion variability to disrupt assessment errors  – in the dopamine ON condition, increases in exertion variability had a less pronounced effect on discrepancies between actual exertions and assessments. We found that healthy-control participants’ assessment error matched participants in the dopamine OFF , and the dopamine ON conditions . These results show that dopamine influences the degree to which exertion variability impacts the transformation of exertion performance into assessments of effort.Overall, participants’ normalized exertion variability was related to the magnitude of their errors in effort assessment in both the Control  shifted in the ON compared to the OFF dopamine conditions. We have previously shown that inflated subjective effort cost valuation was accompanied by increased risk aversion for prospective effort20. Consistent with both effort assessments and prospective valuations of effort having lower aversive cost in conditions of higher dopamine availability, we found that participants were less risk averse for effort in the dopamine ON condition compared to OFF . Since each effort choice was presented twice (ON condition and OFF condition), we could also examine the relationship between risk preference for identical choices. This analysis further confirmed that participants were less risk averse for effort in the dopamine ON condition compared to OFF . We also had participants perform a risky monetary decision-making task . These results suggest that dopamine’s influence on risk preferences was specific to effort-based decision-making.Finally, if increased dopamine availability is related to judgments of effort, we would expect that dopamine may also have an influence on participants’ prospective decisions about effort. To test this hypothesis, we had participants perform an effort-based decision-making task, in isolation from reward, in both the ON and OFF dopamine conditions. During this task, participants made choices between a low amount of prospective effort with certainty; or an option involving either a prospective high-effort exertion or no exertion, with equal probability Fig. 19–21. Thask Fig. , separat22 and could be related to increased exertion variability. 12 of 19 participants in our study were classified as tremor dominant using their UPDRS scores. To test if increased exertion variability was generally related to dopaminergic modulation of Parkinsonian tremor, we evaluated participants’ exertion variability during the association phase of the experiment. We found that while exertion variability increased in proportion to mean exertion in the association phase , this variability was far lower than in the assessment phase, and the relationship between mean exertion and variability was not influenced by dopamine supplements in the association phase . This illustrates that the dopaminergic modulation of exertion variability observed in the assessment phase was specific to the context of effort assessment and not generally related to the effect of Parkinsonian symptoms on motor performance. We also tested if dopamine had an influence on overall motor capacity by testing if there was a difference between participants’ MVC in the ON and OFF conditions and found no differences . These control analyses suggest that dopaminergic differences in effort assessment were not simply the result of the influences of dopamine on effort association or overall motor capacity, but rather on the performance of effort assessments specifically.It is important to note that tremor (involuntary oscillation of a limb about a joint) is one of the most common symptoms of PDn = 19, r = 0.16, p = 0.51) and exertion variability . We did not find significant relationships between dopamine-induced changes in behavior and clinical measures of Parkinson’s disease state.We also tested if there were relationships between UPDRS scores and changes in the relationships between parameter estimates with effort assessment and mean exertion  or within individual target effort levels .To confirm that the treatment effects observed in the assessment phase were not solely a byproduct of the influence of dopamine on the development of associations between levels of exertion and units of effort, we analyzed participants’ performance during the association phase. We did not find a significant influence of dopamine availability on success/failure during the association phase, either overall . To confirm that the dopaminergic effects on effort assessments we observed were not simply the result of differences in exertion ramp time, we performed a regression between participants exertion ramp times and effort assessments. We did not find a significant interaction between ramp times in the ON and OFF dopamine conditions and effort assessments . These findings suggest that exertion ramp time alone did not influence effort assessments.A previous study of motor and motivational contributions of dopamine to exertion and decision found that increased dopamine availability was associated with a decreased time to reach peak exertion when compared to a dopamine-depleted state25 and experimental12 accounts that have suggested that dopamine availability influences features of motor performance and facilitates the more salient encoding of stimuli. Our finding of improved accuracy in effort assessment in conditions of increased dopamine availability aligns with previous studies of effort-based choice, which found that dopamine increased the likelihood of selecting high-effort options, consistent with decreased levels of effort aversion27. However, these previous studies focused on dopaminergic function during stimulus discrimination and effort/reward trade-offs and did not consider how dopamine impacted the more basic functions of subjective effort assessment. Our results go beyond these studies by providing a framework within which dopamine influences fundamental parameters of exertion , and higher-level cognitive assessments of effort. While previous work has shown that dopamine mitigates effort cost through incentivization of reward, here we demonstrate the role of dopamine in influencing assessments of effort and associated effort-based decision-making.Here we show that increased dopamine availability reduces variability in effortful exertion and that these decreases in variability are related to increased accuracy of effort assessment. These findings show that dopamine has a protective effect on assessments of effort by reducing variability in motor performance. Our results align with previous theoretical29, and negative effects by reducing the accuracy of intended movements30. Movement while in a dopamine-depleted state is more variable, especially as the magnitude of the required movement increases32. Here we demonstrate that increased motor variability, within the framework of sustained force production, influences assessments of effort. We did not observe dopamine-modulated differences in exertion variability with effort when the target effort was explicitly presented to participants in the association phase. This suggests that the differences during the effort assessment phase may arise from dopamine’s influence on the transfer of movement parameters to assessments of effort. Uncertainty arising from this variability may affect the judgment of task performance, leading to inflations in assessment. We have recently showed, in healthy participants, that increased variability serves as an added cost that inflates assessments and valuation of effort13.Motor variability can have positive effects on motor learning by facilitating exploration and improving learning6. In this framework, it is possible that dopamine directly effects subjective assessments of exerted physical effort, which influences risk preferences for prospective decisions about effort exertion.Increased feelings of effort are likely to influence the boundary between effort-reward trade-offs, such that the reduced willingness to exert for reward in a dopamine-depleted state may be driven by inflated estimates of effort valuation in addition to decreases in reward sensitivity. Our finding that dopamine depletion increases variability in motor performance and risk aversion for effort, isolated from reward, aligns with previous findings of an increased reluctance to allocate physical effort in individuals with PDAnother potential interpretation of our data is that dopamine availability could influence the accuracy with which an individual exerts effort, making exertions feel more difficult, and in turn, effort more costly. If this were the case dopamine availably would directly impact the efficiency of the motor system, to eventually influence effort assessments. Our experimental paradigm was not designed to differentiate between dopamine’s influence on effort exertion, assessment, or an interaction between the two. In the future it will be important to design studies of effort assessment that are able to dissociate the influence of dopamine on these intertwined variables.33. We did not find a significant relationship between dopamine and performance during the association phase, and the behavioral effects of dopamine availability were best described by effort assessment performance. However, our experiment was not specifically designed to dissociate effort-based learning and valuations of effort. Experiments that explore individuals’ assessments of effort throughout the course of effort-based learning could elucidate the interactions between the generation of effort associations and valuation.Errors in effort assessment could arise from inaccurate estimates of exerted effort, or from an incorrect mapping between force production and effort levels during association. It has been shown that when learning about effort/reward trade-offs, reward and effort prediction errors are commonly encoded in the dopaminergic midbrain, suggesting a common neural substrate that underlies the generation of effort and reward associationsIt is also important to note that when comparing data from the control and PD groups, we only found a significant difference between the control group and participants in the ON condition, in the effort assessment analysis. The lack of significance in the other analyses was likely due to our experiment being underpowered to detect significant differences between the control and patient groups, while comparisons between the ON and OFF dopamine conditions relied on more statistically powerful pairwise analyses. Essentially, it was difficult to detect significant differences between the control and PD groups because we were making population level comparisons between groups, while between the PD ON and OFF conditions we were able to use within participant comparisons.In summary, our study shows that dopamine availability influences the transformation of physical exertion into assessments of effort by modulating the extent to which exertion variability impacts judgments of effort. This work begins to bridge the gap in understanding how dopamine influences the translation of motor performance into assessments of physical effort.The protocol was approved by the Johns Hopkins University Institutional Review Board, and all participants provided written informed consent. A total of 24 persons with Parkinson’s disease participated in this study and were pre-screened to exclude those with any other neurological disorders. Five participants were excluded from the analysis for a variety of reasons. One participant did not express willingness to return to complete the study, one participant reported having an implanted deep brain stimulator (only after the first session was completed), and one participant had pronounced tremor that interfered with the ability to control a mouse to report exerted effort, and two participants had severe cognitive difficulties and were unable to fully follow the experiment instructions. The final cohort was comprised of a total of 19 participants  and were asked to assess the level of exerted effort using a sliding scale presented on a computer screen in front of them. During experiments, signals from this dynamometer were sent to our software for real-time visual feedback of participants’ exertion. To record participants’ assessments of effort and choices, we collected keystrokes from a standard computer keyboard. Exertions with the dynamometer were performed with participants’ dominant hand, and keyboard selections were made with the nondominant hand.Participants were presented with visual cues using custom MATLAB (Mathworks 2018a) code utilizing PsychToolBox libraries20.Before beginning the experiment, participants were told that they would receive a show-up fee of $15/h, and that this fee was not dependent on their performance over the course of the experiment. The effort assessment paradigm was identical to those we have previously usedOn each day of the experiment participants began by exerting their maximum voluntary contraction (MVC). Participants were asked to squeeze the dynamometer as hard as they could for four seconds, on three consecutive trials, and each trial was followed by a period of rest. A participant’s MVC was defined as the maximum force exerted over all trials.Participants were next evaluated in an association phase where they were trained to associate the force they exerted against the dynamometer with different levels of effort Fig. . Effort Participants next performed an effort assessment phase to determine how they generated assessments of their levels of exertion, and if dopamine influenced these assessments. Participants were tested on each of the previously trained effort levels . Each assessment trial began with a black horizontal effort gauge that participants were instructed to fill completely, and hold, by exerting on the force transducer. Participants were given 4 s to exert, and as for the association phase, instructed to squeeze to reach the end of the gauge as quickly as possible and sustain the effort level for the duration of the task (4 s). Unlike the association phase, the end of the effort gauge did not represent effort level 100. Instead, it represented the target effort level selected from the range 10–80 effort units, incremented by 10 effort units. In this way participants were not given explicit feedback of the units of effort associated with their exertion. Following exertion, participants were presented with a number line that ranged from 0 to 100 and instructed to select the effort level that they believed that they had just exerted. The cursor was moved along a number line using a mouse to change the value and clicking the left mouse button to select an effort level. Participants were tested on 48 trials, with 6 trials for each effort level from 10 to 80. These effort levels were presented in a random order, and no feedback was provided to the participants about the accuracy of their effort assessments. Upon selection, a fixation cross appeared on the screen for 1–4 s to provide rest between trials. A longer rest of 20 s was provided midway through the phase.21 Before being presented with the effort gambles, participants were told that 5 of their decisions would be selected at random at the end of the experiment and that they would have to remain in the testing area until they achieved the exertions required. This was done to ensure that participants were properly incentivized on each trial.After the assessment phase, participants performed a series of effort gambles and the choices from these gambles were used to characterize participants’ risk preferences and associated subjective preferences for effort. We have used this paradigm in a series of previous studies fs) with certainty; and a risky option that could result in either high exertion (fg) or no exertion, with equal probability : exerting a low amount of force , which allowed us to isolate processes related to effort valuation, separate from the effects of reward valuation or effort/reward trade-offs.This effort choice paradigm exploits the theoretical equivalence between risk preferences and subjective valuation to measure subjective valuation via the presentation of risky choices, a widely accepted practice in economics and decision neuroscienceParticipants with PD performed the effort choice task on both days of the experiment, and those in the control group performed the effort choice task once.39.At the end of the experiment participants were also tested on their monetary risk attitude using a series of gambles that included only financial gains. In each trial, each participant was presented with the choice either to accept a safe option  or to play a risky gamble . The sure amount ranged from $1 to $12. Corresponding gambles ranged from $2 to $30. A total of 20 trials were presented. At the end of the experiment a trial was randomly selected, and a payment was made according to the participants’ decision and a random outcome. These exact choices have been used to elicit risk aversion in several previous studiesTo analyze participants’ exertion performance during the assessment and association phases we calculated metrics of performance for the final 3 s of the 4 s exertion segment (henceforth referred to as the target period). We excluded data from the first second of trials to remove variability in performance arising from different response times. Exertion performance metrics were expressed in effort units.ME) during the target period. To evaluate exertion variability (EV) we calculated the standard deviation of participants’ exertion during the target period. We also calculated a normalized exertion variability (EVN) metric in which we divided the standard deviation of participants’ exertion during the target period by their mean exertion . The normalized variability controlled for the relationship between increasing levels of exertion and exertion variability so that we could evaluate how trial-to-trial variations in exertion variability were related to assessments of effort.We calculated participants’ mean exertion . When comparing treatment conditions (Dopamine ON vs OFF), we used a mixed effects model with participant as a random effect, and the treatment condition, appropriate covariate , and the interaction between these factors as fixed effects. When comparing two populations , we used a mixed effects model with participant as a random effect nested under population, and fixed factors as described above.We used mixed-effect linear models to assess the relationship between participants’ trail-to-trial variance in exertion performance was related to trial-to-trial variance in their ratings of exertion, and how dopamine influenced these relationships. These analyses were performed using MatabR2022a, using the i denoted trial number, j denoted subject number (1–19), and Treatment was a factorial variable . This analysis was performed for both the Association and Assessment phases.To evaluate the accuracy of participants assessment of effort we computed the above model individually for each subject, in each treatment condition, and compared the population slope to using a single sample t-test unity (perfect effort assessment would correspond to a slope of 1).EVN). This model allowed us to test if variability in exertion contributed to participants errors in effort assessment.We also computed a model to test how trial-to-trial discrepancies between participants’ effort assessments and their mean exertion  were related to their normalized exertion variability  who had an acceptance rate below 5% or greater than 95% as we took this to indicate a lack of evaluating effort options, and merely choosing the same option on every trial. We calculated the monetary choice similarity metric for the same group of participants as for the effort similarity metric.To assess how risk attitudes  for effort and money change between treatment conditions, we compared choices between conditions by computing a choice similarity metric. This metric is model-free and does not assume an effort utility function or require the fitting of a model to the behavioral data. Since each effort/monetary gamble was presented twice (once per treatment condition), it is possible to examine if choice behavior for identical effort options changed between treatment conditions. To generate this metric, a value of 0 was assigned to a choice trial in the ON condition if the participant made the same choice as in the OFF condition; +1 was assigned to a choice if the participant accepted a gamble in the ON condition that they rejected in the OFF condition ; and −1 was assigned if the participant rejected a gamble in the ON condition that they accepted in the OFF condition . In the analysis of effort choice, we excluded participants (Further information on research design is available in the Supplemental MaterialReporting Summary


Density functional theory calculations predicted weak EPC strength, λ, supporting an unconventional pairing mechanism in CsV3Sb5. However, experimental determination of λ is still missing, hindering a microscopic understanding of the intertwined ground state of CsV3Sb5. Here, using 7-eV laser-based angle-resolved photoemission spectroscopy and Eliashberg function analysis, we determine an intermediate λ=0.45–0.6 at T = 6 K for both Sb 5p and V 3d electronic bands, which can support a conventional superconducting transition temperature on the same magnitude of experimental value in CsV3Sb5. Remarkably, the EPC on the V 3d-band enhances to λ~0.75 as the superconducting transition temperature elevated to 4.4 K in Cs(V0.93Nb0.07)3Sb5. Our results provide an important clue to understand the pairing mechanism in the kagome superconductor CsV3Sb5.In crystalline materials, electron-phonon coupling (EPC) is a ubiquitous many-body interaction that drives conventional Bardeen-Cooper-Schrieffer superconductivity. Recently, in a new kagome metal CsV 3Sb5, but an experimental measurement is lacking. Here, the authors use ARPES measurements to find that electron-phonon coupling in CsV3Sb5 is strong enough to support the experimental superconducting transition.Electron-phonon coupling is thought to be too weak to be responsible for the superconducting Cooper pairing of the kagome metals AV Due to the wavefunction interference, the electronic structure of the kagome lattice features flat band, Dirac fermion, and van Hove singularities that result in a rich interplay between topology, geometry, and correlations5. For kagome metals near the van Hove singularities, the high density of states combining with the frustrated lattice geometry are predicted to support novel electronic orders8. Recently, in a topological kagome metal CsV3Sb5, superconductivity that possibly intertwines with charge density wave (CDW)12  studies20 observed multiple van Hove singularities from V 3d-electrons near the Fermi level (EF), highlighting electronic driven instabilities8  by determining the EPC-induced kinks in the electronic band structure. Our results reveal an intermediate EPC with λ=0.45–0.6 in CsV3Sb5, which can support a Tc on the same magnitude of the experimental value. Intriguingly, we find that λd is enhanced by about 50% in the isovalent-substituted Cs(V0.93Nb0.07)3Sb5 with an elevated Tc = 4.4 K. Our results suggest that EPC can play an important role on the superconductivity in CsV3Sb5.The kagome lattice, made of corner-shared triangles, is an exciting platform for emergent quantum phenomena)12 Fig. , nematices8 Fig. . Further3Sb5, respectively. In agreement with previous DFT and ARPES studies21, the Sb 5p-band forms a circular FS, marked as α, at the BZ center and the V 3d bands yield hexagonal and triangle FSs, marked as β and δ in Fig. 24, near a binding energy EB~32 meV. This many-body effect can be quantified by fitting the ARPES momentum distribution curves (MDCs) with a Lorentzian function25:E-EF) and ImΣ(ω = E-EF) are the real and imaginary parts of the single-particle self-energy. ε(k) is the non-interacting bare band that can be approximated as a liner dispersion crossing EF23. Figure 26  and a step jump in ImΣ(ω) prove strong many-body interactions. Since the self-energy anomalies persist above CDW transition temperature TCDW  of the α band only shows a broad shoulder.Figure and Fig. . The 12-p and 3d bands points to a non-neglectable role of EPC for superconductivity in CsV3Sb5. To test the EPC-driven superconductivity, we extract the Eliashberg function, T = 6 K, slightly above Tc, using the maximum entropy method28 (see methods). A fit of the ReΣ(ω) and the extracted 29:α2F(ω), where phonon modes near 32 meV are accounted for 70% of the total EPC strength on the α band, λp, but less than 50% for the EPC strength on the β band, λd. Interestingly, due to the spectral weight redistribution in p and λd are similar with λp,d~0.45 ± 0.05. We also employed the MEM fits the extracted ImΣ(ω), which yields a λ cοnsistent with the ReΣ(ω) fits . Using McMillan’s formula30 and taking the lower and upper limits of the experimentally estimated Tc in a range from 0.8 K to 3 K 3Sb5 with Tc~4.4 K34. Electronic Kinks are observed on both the α and β bands as shown in Fig. 0.93Nb0.07)3Sb5. The shaded area corresponds to the ReΣ(ω) of the pristine CsV3Sb5. Remarkably, we observe that while ReΣ(ω) on the α band is similar in Cs(V0.93Nb0.07)3Sb5 and CsV3Sb5, on the β band, it shows a strong enhancement in the Nb-doped sample, especially near EB~10 meV. Based on the extracted α2F(ω), shown in Fig. d~0.75 ± 0.05 is enhanced by about 50% in Cs(V0.93Nb0.07)3Sb5 . Such giant enhancement is also manifested by the slope of ReΣ(ω) near EF 3Sb5 is expected to elevate Tc up to 4.5 K  and ImΣ(ω) linked by Kramers-Kronig transformation26 validates the linear bare band assumption for the α and β bands 3Sb5. Our results provide an important clue to understand the pairing mechanism in CsV3Sb5. The orbital, momentum of electronic kinks and their strengthening with the promoted Tc prove that the EPC in CsV3Sb5 is strong enough to support a Tc comparable to the experiment value and hence cannot be excluded as a possible pairing mechanism. While the exact microscopic pairing mechanism calls for further scrutiny, it is important to point out that the EPC-driven superconductivity is not incompatible with the recently observed pair-density wave (PDW) in CsV3Sb517. Indeed, PDW has been observed in another conventional superconductor NbSe2, where the pair-density modulation is due to the real space charge density modulations36. We also note that the EPC-driven superconductivity can coexist with the time-reversal symmetry-breaking (TRSB) orders or fluctuations38, as proposed by theoretical studies41. In those cases, the superconducting order parameter is expected to intertwine with the TRSB order parameter, which gives rise to an unconventional ground state.In summary, by investigating the electronic kinks, we determined an intermediate EPC that is twice larger than the DFT calculated value in the kagome superconductor CsV3Sb5 were grown using CsSb2 alloy and Sb as flux. Cs, V, Sb elements and CsSb2 precursor were sealed in a Ta crucible in a molar ratio of 1:3:14:10, which was finally sealed in a highly evacuated quartz tube. The tube was heated up to 1273 K, maintained for 20 hours and then cooled down to 763 K slowly. Single crystals were separated from the flux by centrifuging. The single crystals of Cs(V0.93Nb0.07)3Sb5 were provided by Jinggong New Materials (Yangzhong) Co., Ltd. The growth and characterizations of Cs(V0.93Nb0.07)3Sb5 were present in ref. 34. The magnetic susceptibility was measured under magnetic field 20 Oe for CsV3Sb5 and 5 Oe for Cs(V0.93Nb0.07)3Sb5.Single crystals of CsV42. The energy resolution was set to be 1.3 meV. The sample temperature was set to be 6 K if there is no special announcement. The samples were cleaved in situ and kept under a vacuum better than 3 × 10−11 torr during the experiments.ARPES measurements were performed for the freshly cleaving surface with a Scienta-Omicron R4000 hemispherical analyzer with an ultraviolet laser (hν = 6.994 eV) at the Institute for Solid State Physics, the University of Tokyo28, which is frequently used to perform the analytic continuation43. By considering the energy resolution of the laser-ARPES, we estimated that the error bar of the real part of the self-energy was 1 meV. MEM requires a model default function to define the entropic prior. Here, we adopted the following model:28.The Eliashberg function Supplementary informationPeer Review File


In this review, we discuss the contributions of high-resolution microscopy studies to enlarge the body of morphofunctional data to understand the biology of the BTB as a dynamic structure. The first morphological evidence of the BTB was based on a fine structure of the junctions, which was resolved with Transmission Electron Microscopy. The use of conventional Fluorescent Light Microscopy to examine labelled molecules emerged as a fundamental technique for elucidating the precise protein localization at the BTB. Then laser-scanning confocal microscopy allowed the study of three-dimensional structures and complexes at the seminiferous epithelium. Several junction proteins, like the transmembrane, scaffold and signaling proteins, were identified in the testis using traditional animal models. BTB morphology was analyzed in different physiological conditions as the spermatocyte movement during meiosis, testis development, and seasonal spermatogenesis, but also structural elements, proteins, and BTB permeability were studied. Under pathological, pharmacological, or pollutant/toxic conditions, there are significant studies that provide high-resolution images which help to understand the dynamic of the BTB. Notwithstanding the advances, further research using new technologies is required to gain information on the BTB. Super-resolution light microscopy is needed to provide new research with high-quality images of targeted molecules at a nanometer-scale resolution. Finally, we highlight research areas that warrant future studies, pinpointing new microscopy approaches and helping to improve our ability to understand this barrier complexity.Blood-testis barrier (BTB) creates a particular compartment in the seminiferous epithelium. Contacting Sertoli cell-Sertoli cell plasma membranes possess specialized junction proteins which present a complex dynamic of formation and dismantling. Thus, these specialized structures facilitate germ cell movement across the BTB. Junctions are constantly rearranged during spermatogenesis while the BTB preserves its barrier function. Imaging methods are essential to studying the dynamic of this sophisticated structure in order to understand its functional morphology. Isolated Sertoli cell cultures cannot represent the multiple interactions of the seminiferous epithelium and  Since the first contributions to the field of spermatogenesis, microscopy-based studies were fundamental to understanding cell composition and associations in the seminiferous epithelium . The spein vitro models can represent the situation in situ where multiple cell types interact synchronously (Blood-testis barrier (BTB) is a complex cell structure present in the seminiferous epithelium. It creates two tissular compartments: the basal one, with spermatogonia and early spermatocytes; and the adluminal one, with spermatocytes and post-meiotic germ cells . BTB wasronously .In the mammalian testis, specific cell associations occur along the seminiferous tubule. Depending on maturation, germ and Sertoli cells are exposed to different microenvironments and physiological conditions. Under these stimuli, BTB junctions are rearranged through a dynamic and precise spatio-temporal process .in situ. Still, the current knowledge of junctions’ dynamics and regulatory factors like cytokines, proteases, and androgens mainly comes from in-vitro models using Sertoli cells. After the refinement of immunolocalization techniques and the setup of Sertoli cell cultures, a substantial number of studies on the Sertoli cell barrier’s functional morphology were based on in vitro models  . Furthero models . Cytologin situ barrier biology. We overview several studies on BTB functional morphology under different physiological and pathological situations. Finally, we discuss future directions in the study of the BTB with new technologies of high-resolution microscopy.Here, we review the contributions of high-resolution microscopy (HRM) to the advancement in the study of BTB cellular and molecular morphology, based on animal model studies that analyze the in situ were considered.Considering the complexity of the seminiferous epithelium, to focus on the barrier function of Sertoli cells in the testis, only the studies regarding the junctional complex at the Sertoli cell–Sertoli cell interphase of the BTB 1. Structural features of the BTBSpermatogonia undergo spermatogenesis evolving to mature spermatids through a complex process, and finally spermatozoa are produced . The stuThe BTB is mainly made up of widespread tight junctions (TJs) between somatic Sertoli cells forming paracellular seals close to the basement membrane  Figure . With sc2. Electron microscopy studiesAlthough desmosome elements have yet to be identified using antibodies , variousBefore immunolocalization, electron microscopy allowed the fine study of the BTB. There are both transmission and scanning microscopy studies, the latter of which have made studying the lateral surface of the Sertoli cell possible.Correlative TEM and freeze-fracture microscopy studies were used to understand the nature of TJ at the BTB, and to distinguish whether a TJ was a focal contact or a seal with a barrier function . The sur3. Localization of single molecules and adhesion protein complexes in the BTBImmunoelectron microscopy carried out in fracture replicas revealed oligodendrocyte-specific protein (OSP)/claudin-11 distributed on the fully formed TJ strands of Sertoli cells  and overIn this section, reports showing high-resolution images of the immunolocalization of structural proteins of the BTB and others that are part of the signaling that regulates the junction complexes and the permeability of the barrier are mentioned.Targeting of single proteins in the testis by immunofluorescence (IF) and immunohistochemistry became a key tool for understanding the architecture of body tissues. After the 90s, massive use of this technique was reported in several publications . Then, lThe TJs in the Sertoli cell membranes embody the structural basis of the BTB. BTB’s TJs are composed of two main transmembrane proteins, claudin-11 and occludin. These integral proteins comprise the skeleton of the TJs and are arranged as particles along junctional strands, making up for only a small portion of the intramembranous particles at the junctional fibrils . AdherenThe TJ structure comprises a transmembrane region with identical transmembrane proteins attached to both sides . BesidesClaudins are the major TJs’ integral transmembrane proteins . The disZonula occludens-1 (ZO-1) is an adaptor protein that links transmembrane proteins to the actin cytoskeleton. The position of the scaffold cytoplasmic protein ZO-1 was well characterized in several studies . It was Focal adhesion kinase (FAK) is a major regulator of the BTB as a non-receptor protein kinase . At the Occludin is an integral membrane protein constitutive of tight junctions. In adult mice and rat testis, occludin is distributed in a linear pattern in the basal regions of the Sertoli cells’ TJ strands. Also, its distribution overlapped with ZO-1 at the basal third of the Sertoli cells .w/Kitwv mice model which received spermatogonial stem cell transplant  showed varying localization according to germ cells’ differentiation stage in a mutant infertile Kitansplant .Beta-catenin is a plaque protein associated with cadherin, found in the basal Sertoli cell cytoplasm .At the cadherin-based junctions, p120 is attached to cadherin in the cytoplasmic domain. In the rat testis, p120 was observed at the basal junctions by immunolocalization, but not associated with ectoplasmic specialization . In the Plectin, a plakin protein. Is found at the basal interface in Sertoli cell-cell junctions. Plectin co-locates with vimentin at the end of the intermediate filament and is distributed as focal patches .N-cadherins are transmembrane adhesion proteins  found inCortactin is an F-actin filament-binding protein linked to intercellular junctions. At the junction, cortactin is associated with the junction proteins N-cadherin and E-cadherin, and accessory proteins like ZO-1, p120-catenin, and β-catenin . In the Another member of the Claudins family, claudin-5, has been reported to contribute to BTB function in mice. It was largely expressed along the different stages of the seminiferous epithelium with strong staining at stage VIII, nearby preleptotene, and leptotene spermatocytes .in situ  and the Coxsackie and adenovirus receptor (CAR), were studied in situ .Filamentous actin (F-Actin), another BTB component of the basal ES, was localized in the seminiferous epithelium and actin dysregulation was observed in a non-collagenous (NC) 1 domain peptide overexpression model. The structure of actin and microtubule cytoskeleton, and the localization of regulatory proteins were analyzed, and alterations were also associated with NC-1 peptide overexpression . CalmoduOther regulators have been also identified. Fyn immunoreactivity was detected in Sertoli cells along the different stages of the seminiferous epithelium, concentrated at the basal sites of the epithelium, where ESs are found .Cell surface proteins like the single transmembrane coxsackievirus and adenovirus receptor (CAR) are present in most epithelial TJs. CAR was found in the basal compartment in all rat testis stages. During fully active spermatogenesis, CAR and ZO-1 co-localized near the base of the seminiferous epithelium, showing that both are constituents of the BTB .Connexin (Cx) 43 is the principal protein of the gap junction (GJ) of the testis. GJs are typical BTB constituents found at the contact membrane between Sertoli cells . At the Two studies showed stage-specific localization proteins involved in actin and microtubule cytoskeleton regulation . Scribblin vitro Sertoli cells’ models4. Current limitations of Intermediate filaments are one of the three major elements of the cell cytoskeleton and are associated with cell junctions at Sertoli-Sertoli cell junctions. The localization of vimentin and other non-typical intermediate filaments as glial fibrillary acid protein (GFAP) and neurofilaments (NF) were targeted in the Sertoli cell cytoplasm . Vimentiin situ . Since the possibility of tracking live events using cell culture and the accuracy of immunolocalization performed on cytological preparations, new models in vitro, including co-culture of Sertoli and germ cells, are needed that more closely relate them with in-vivo BTB models.5. BTB junction assembly during testis developmentIn preparations using an appropriate substrate, Sertoli cells maintain morphological characteristics and establish TJ complexes similar to those present in situ . Transepid cells . The higed alone . Considepostpartum (dpp)  . GJ protum (dpp) .During development and tissular morphogenesis, CAR is present at high levels, mediating cell adhesion . Few stuThe BTB assembly during puberty was studied in 14–20 dpp using TEM and permeability tracers, showing an association between the absence of the BTB and the apoptosis of zygotene-pachytene spermatocytes .6. BTB and transcellular germ cell migrationOccludin was targeted in embryonic and developing mouse testis showing a diffuse localization in embryonic cords and a reorganization at the basal junction of postnatal prepuberal individuals, showing the initial TJ layout .6. BTB a7. BTB and the controversy about the intermediate compartmentThe transfer of spermatocytes from one compartment  of the seminiferous epithelium to another  is necessary for germ cell maturation. In the adluminal compartment, meiosis is completed, and spermatids formation is followed by spermatid differentiation and spermatozoa release . The pro8. Junction assembly and dismantling in seasonal breedersMorphological studies reveal a third compartment in the seminiferous epithelium . At the Testosterone regulates cell adhesion proteins in the seminiferous tubules . In seasLagostomus maximus), a seasonal reproducer that presents no BTB competence during the inactive period, as confirmed using permeability tracers , different proteins of the Sertoli cell junctions were analyzed during the active phase of spermatogenesis over the different stages of the seminiferous epithelium and in the regressive testis phase , structural markers of the BTB, like claudin-11, Cx43, E-cadherin, and N-cadherin, were studied. BTB was compromised after testis regression, as shown using permeability tracers and inferred by TEM (Crocidura russula), claudin-11 was mapped during the active and regression phases of spermatogenesis (Hemiechinus auritus), with a disruption of tight and gap junctions as shown by claudin-11 and Cx43, respectively. Adherens junction molecules, N-cadherin and β-catenin, were also targeted by IF . Humans are exposed to Cd through pollutants in the air, drinking water, and food, and Cd impairs Sertoli cell development during fetal and neonatal phases . On geste testis . BTB mare testis . Cadmiume testis  and altee testis .in vitro” studies, 0.5 μm–10 μm MPs were internalized by the germ, Leydig, and Sertoli cells , emerging pollutant particles smaller than 5 mm, have been reported to induce testicular disorder in mammals . In “in li cells , disruptli cells . Likewises level . Polystyes level . Oral tres level . The simes level .12. Permin situ using electron or light microscopy. Lanthanum nitrate and horseradish peroxidase are the most widely used in TEM. Lanthanum has the disadvantage of forming precipitates responsible for false positives and unreliable staining . Super-resolution microscopy accuracy allows molecular localization within the nanoscale range, which is required to understand the significant diversity of protein interactions in the BTB  in situ .Three-dimensional structures like cells, organelles, and the ultrastructure of the cellular membrane can be examined using confocal microscopy  and ElecFine-resolution techniques have been used to analyze Sertoli cell organelles in rat testis. Using Electron tomography, the 3D aspect of tubulobulbar complexes was described . Basal EAlso, using Fluorescent Labeling of Abundant Reactive Entities (FLARE) and expansion super-resolution microscopy, spermatid development was imaged showing specific carbohydrate patterns . In contin situ. Imaging the dynamic nature of the junctions that seal the adluminal space is a challenge for morphological studies. Therefore, visualization of specific structures and interactions of the junctions at a high spatial resolution is necessary to improve the actual knowledge of the BTB. Further studies using microscopic techniques like electron tomography and confocal microscopy, which ponder the three-dimensional junction structures, are yet to be done. The junctions at the BTB are one of the largest obstacles to male contraception, since many drugs cannot reach the adluminal compartment. Addressing BTB functional morphology issues will help to understand how it is regulated and design new strategies for male fertility control and contraception.The blood-testis barrier has a central role in spermatogenesis regulation. So far, the classical ultrastructural and IF research framework has supported BTB functional morphology. As observed in a standard cross-section of the testis, there’s a great diversity of cell associations that occur between cells. During spermatogenesis, BTB needs to keep a safe and isolated environment for postmeiotic germ cell maturation. Because of its complex structure, the BTB can be fully understood only considering the whole seminiferous epithelium