Although there is evidence that Attention Deficit Hyperactivity Disorder (ADHD) symptoms are positively related to alcohol use and related problems among young adults, little research has examined the mechanisms that might explain this association. In response, this study examined the mediating effects of coping and enhancement drinking motives on the prospective associations between ADHD symptoms and alcohol outcomes.N = 4,536) were young men from the Cohort Study on Substance Use Risk Factors. Measures of ADHD symptoms and those of drinking motives, heavy episodic drinking (HED) and alcohol use disorder symptoms were used from the baseline and 15-month follow-up assessments.Participants  symptoms and with HED were partially and completely mediated through drinking motives, respectively, whereas drinking motives did not mediate the ADHD-hyperactivity/impulsivity-symptoms-alcohol outcomes associations.Results indicated that coping and enhancement motives partially explained the ADHD-inattention symptoms—subsequent alcohol outcomes association. These findings suggest that interventions targeting enhancement and coping motives may help prevent problematic drinking among young men with elevated ADHD-inattention symptoms. Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder characterized by inattentive  and/or hyperactive-impulsive symptoms  affecting 5–10% of school-age children . ContrarThere is also evidence that ADHD is a correlate of problematic drinking in adults , 15. NotIncreasing evidence suggests that ADHD stands at the end of a continuum and that ADHD symptoms may occur in the absence of the full disorder , 23. AccRecent research has started to examine the mechanisms through which ADHD may promote alcohol outcomes. That said most studies examining this line of research has focused on the associations between childhood ADHD and subsequent AUD. For instance, a study conducted in the general adult population found that the positive association between childhood ADHD and future AUD was mediated by conduct disorders . In anotTo the best of the authors’ knowledge, only three studies to date have attempted to identify factors that may explain the associations between ADHD symptoms and alcohol outcomes in samples of non-clinical adults. The two first studies were conducted among college students and identified disinhibition, difficulty stopping drinking, lack of premeditation and sensation seeking as significant mediators of the positive association between ADHD  and current alcohol use and/or related consequences , 31. TheOther correlates of drinking that may play a role in the ADHD symptoms-alcohol outcomes association include drinking motives, which are considered among the most proximal factors for engaging in drinking behaviors , 34. AccSimilarly, it is likely that individuals with elevated ADHD symptoms endorse high enhancement drinking motives; indeed, considering that two core ADHD symptoms include being easily bored and having aversion of delayed reward, alcohol use may represent a means of reducing boredom feelings by providing immediate stimulation . EchoingAs far as we are aware of, the mediating effect of coping and enhancement drinking motives on the association between ADHD symptoms and alcohol outcomes in adults has not been tested yet. As outlined below, very little research to date has explored mediators of the association between ADHD symptoms and alcohol outcomes in adults. Answering a call to conduct more research examining potential explanatory factors of this association in large samples , this stBased on previous research, we expected that ADHD symptoms would be positively related to subsequent alcohol outcomes. We also expected that these associations would be partially mediated by both drinking motives with models testing ADHD-HI symptoms being mediated by enhancement drinking motives and those testing ADHD-I by coping drinking motives.This study used data from the Cohort Study on Substance Use Risk Factors designed to examine substance-use trajectories among young Swiss men. Recruitment took place between August 2010 and July 2011 in three of a total of six recruitment centers covering 21 of the 26 Swiss cantons (including French and German-speaking cantons). In Switzerland, all Swiss men aged approximately 19 must undergo a recruitment process to determine their eligibility for military service. All young men were eligible for inclusion, independently of whether they were deemed eligible or not to serve in the army. Thus, virtually all men aged 19–20 in the 21 covered cantons were eligible for inclusion in the study. Participants completed questionnaires outside of the army environment. At baseline, conscripts who gave consent to participate were invited by email (or by mail if they asked so) within two weeks to complete a paper pencil questionnaire or an online questionnaire. Then, they received an email (or a mail if they preferred) to complete the follow-up assessment using an online questionnaire . Participants received a voucher after completion of each survey . More details regarding the parent study procedures are provided elsewhere . In the n = 568, 10.4%) were not included in the current study because the questionnaire assessing drinking motives was administrated among 12-month drinkers only. Missing values  on key variables were listwise deleted, resulting in a final sample of 4,536 participants (see SD = 1.19). More than half of the sample was French-speaking (54.30%). Primary school was the most commonly reported highest level of education completed (48.80%), followed by secondary school (28.30%) and tertiary school (22.80%).A total of 7,556 conscripts provided written informed consent to participate in the study. Of those, 5,987 (79.2%) completed the baseline assessment between September 2010 and March 2012, and among them 5,479 (91.5% of the baseline sample) completed the follow-up assessment between March 2012 and January 2014. Non-response analysis indicated that non-respondents reported more alcohol use than respondents. The magnitude of these differences was, however, small, indicating a small non-response bias . For insants see . At baseAge, linguistic region  and highest achieved education were assessed at baseline and served to describe the sample and as covariates in the analyses.How often do you have problems remembering appointments or obligations) and a 2-item hyperactivity/impulsivity subscale . The ASa motor; ). ADHD-tto forget about your problems) or enhancement  motives, using a 5-point Likert scale. Coping (α = 0.84) and enhancement (α = 0.82) drinking motives subscales showed adequate consistency. Both drinking motives were treated as latent variables for ordinal data in the analyses and served as mediators in the analyses.Coping and enhancement drinking motives were measured at 15 months with two 3-item subscales of the Drinking Motives Questionnaire Revised Short Form . Particyou often found yourself drinking more and for longer periods of time than you intended). Reliability and validity of this measure have been supported in previous research ).research , 51. In feeling lacking in energy and strength [Depression symptoms were assessed at baseline with the Major Depression Inventory , which strength . ParticiBefore you were 15 years old, how often did you start fights or bully, threaten, or intimidate others?) and in six others since they were 15 —using a 6-point Likert-scale ranging from 1 to 6. Answers were summed up to yield a total score (ASPD symptoms), which served as covariate in the analyses. Internal consistency was adequate (α = 0.83).The Mini International Neuropsychiatric interview  was usec’) between the predictor  and the outcome , the paths (a) between the predictor and the mediators  and the paths (b) between the mediators and the outcome. Specific indirect associations of the predictor on the outcome through a specific mediator is the product of the paths linking the predictor to the given mediator (a) and the one linking that moderator to the outcome b); the total indirect associations of a predictor is the sum of all specific indirect associations, whereas the total association is the sum of the direct association (c’) and the total indirect association. SEMs show partial mediation when both indirect and direct associations are significant and full mediation when the indirect association is significant, whereas the direct association is not.Four structural equation models (SEMs) were conducted to examine the path from ADHD symptoms to alcohol outcomes through coping and enhancement drinking motives. Two SEMs examined the mediating effects of drinking motives on the associations between ADHD-total symptoms at baseline and a) AUD symptoms and b) HED at 15 months; two other SEMs tested the mediating effects of drinking motives on the associations of ADHD-I and ADHD-HI symptoms at baseline with c) AUD symptoms and d) HED at 15 months. To test mediation , whereas ADHD-I symptoms were significantly related to more AUD symptoms but not to HED. These results are incongruent with some of previous research conducted in young adults that revealed ADHD-HI symptoms to be significantly related to alcohol use , 32 and Our findings notably add to the existing literature by providing initial evidence that the association between ADHD symptoms and subsequent alcohol outcomes may be partially explained by coping and enhancement drinking motives. As expected, findings revealed indirect effects of enhancement and coping drinking motives on the associations between ADHD-total symptoms and subsequent HED and AUD symptoms. Further, findings revealed indirect effects through coping drinking motives on the association between ADHD-I symptoms and subsequent alcohol outcomes. Unexpectedly, findings also revealed significant indirect effects through enhancement drinking motives on the longitudinal association between ADHD-I symptoms and alcohol outcomes. It may be that impairments of executive functioning, such as inattention and distractibility, lead to poor risk perception and impaired ability to deploy appropriate judgment, which may in turn increase the likelihood of engaging in risky behaviors aiming to seek sensation . Past research has documented that adults with ADHD are prone to risky decision making  and, relTaken together, the findings of this study suggest that young adults with elevated ADHD-I symptoms may be more at risk from engaging in problematic drinking because they use alcohol for enhancement and/or coping motives. If replicated by future research, these findings provide important insight regarding the development of alcohol-related interventions tailored to young men endorsing elevated ADHD-I symptoms. Specifically, they suggest that alcohol-related cognitive behavioral interventions targeting both enhancement and coping drinking motives may be a promising way to prevent problematic drinking among young adults with elevated ADHD-I symptoms . Such inAlthough the major strengths of this study include its longitudinal design and its large sample size, findings should be interpreted in the light of several limitations. First, the sample was limited to young males, which precludes generalizability of findings to young females or to other age groups. Second, the study relied on responses to self-reported questionnaires and their validity may be a concern, although participants were assured confidentiality. Third, as mentioned earlier, ADHD-HI symptoms were measured with a 2-item subscale. Although the ASRS-v1.1 has been found to have good psychometric properties , 68, futDespite these limitations, this study makes an interesting contribution to the literature by providing initial evidence that the association between ADHD-I symptoms and future alcohol outcomes is partially mediated by coping and enhancement drinking motives. Although future research is needed to confirm these findings and ensure their generalizability, they suggest that cognitive-behavioral interventions targeting enhancement and coping drinking motives may represent a promising way to decrease alcohol-related harm among at-risk young adults with elevated ADHD-I symptoms.


Echinolittorina malaccana, we investigated heat tolerance plasticity in terms of laboratory acclimation and natural acclimatization of populations from thermally-dissimilar nearby shorelines. Laboratory treatments yielded similar capacities of snails from either population to acclimate their lethal thermal limit (LT50 variation was ∼2°C). However, the populations differed in the temperature range over which acclimatory adjustments could be made; LT50 plasticity occurred over a higher temperature range in the warm-shore snails compared to the cool-shore snails, giving an overall acclimation capacity for the populations combined of 2.9°C. In addition to confirming significant heat tolerance plasticity in tropical intertidal animals, these findings reveal two plasticity forms, reversible (laboratory acclimation) and non-reversible (population or shoreline specific) plasticity. The plasticity forms should account for different spatiotemporal scales of the environmental temperature variation; reversible plasticity for daily and tidal variations in microhabitat temperature and non-reversible plasticity for lifelong, shoreline temperature conditions. Non-reversible heat tolerance plasticity, likely established after larvae settle on the shore, should be energetically beneficial in preventing heat shock protein overexpression, but also should facilitate widespread colonization of coasts that support thermally-diverse shorelines. This first demonstration of different plasticity forms in benthic intertidal animals supports the hypothesis that habitat heterogeneity (irrespective of latitude) drives thermal plasticity selection. It further suggests that studies not making reference to different spatial scales of thermal heterogeneity, nor seeking how these may drive different thermal plasticity forms, risk misinterpreting ectothermic responses to environmental warming.The theory for thermal plasticity of tropical ectotherms has centered on terrestrial and open-water marine animals which experience reduced variation in diurnal and seasonal temperatures, conditions constraining plasticity selection. Tropical marine intertidal animals, however, experience complex habitat thermal heterogeneity, circumstances encouraging thermal plasticity selection. Using the tropical rocky-intertidal gastropod,  In the context of climate warming, a complex theory for this plasticity has emerged, which considers its energetic benefits, environmental drivers and evolutionary constraints  plasticity selection  was investigated from laboratory acclimation experiments. Non-reversible plasticity was assessed by comparing the thermal bands for lethal temperature acclimation of snail populations from warmer or cooler shorelines. Because reversible laboratory acclimation is expected to eliminate the effects of recent field temperature exposures, non-reversible acclimatization was assumed in cases where the thermal acclimation bands varied between the populations.The present study aimed to determine whether the heat tolerance plasticity of Echinolittorina malaccana (Philippi 1847) occurs abundantly on rocky-shores throughout the Indo-Pacific , between 21 June and 22 July 2018  were collected from both shorelines within the proximity of the temperature loggers, while awash and feeding. In the laboratory they were rinsed in freshly-collected seawater to rehydrate the snails before starting the acclimation treatments. Prior to field-fresh thermal tolerance determinations, snails were exposed to blown air  to inactivate them, dry shells and induce withdrawal into the shell . Field-fE. malacccana (Four (4) primary laboratory temperature acclimation treatments were conducted on both warm-shore  and cool-shore  snails. The first set of experiments involved using a programmable Memmert Peltier-cooled (IPP400) incubator to cool-acclimate (CA) one group of randomly selected snails in air at 22–23°C, which approximates the coolest thermal regimes naturally experienced by tropical populations of lacccana . A seconlacccana . Notablylacccana . Anotherlacccana . We furt50; max), the temperature at which neuromuscular co-ordination fails, which is commonly used in ectotherm experiments, is unsuitable for determining gastropod lethality. This is because inactivity in high-shore littorinid snails relates to desiccation-risk-avoidance, and snails with shells glued to rock surfaces withstand temperatures well above those limiting foot physiological performance . Lethality (LT50) was determined for groups of 10 snails that were randomly removed from the water bath at 1°C intervals between 55 and 60°C, and allowed to recover at 28°C in wetted Petri dishes. Snails that emerged from their shells, extended their foot, and remained attached to the surface after 12 h were scored as alive. Alive but unattached snails, which are ecologically non-functional and vulnerable, were scored dead. All experiments were repeated either three or six times based on logistic constraints, with numbers of individuals limited for conservation purposes. One thousand nine hundred individual snails were used in the experiments. The study was approved by the Faculty of Science Ethics Committee, Universiti Brunei Darussalam. The effect of acclimation on LT50 was statistically compared between paired treatments using Generalized Linear Models (GLZM) for a binomial distribution, with a logit-link function . Actual values of LT50 were computed from three parameter logistic regressions, which were plotted using Sigmaplot v14 .To determine heat tolerance, acclimated snails were placed in dry 50 ml glass tubes in a programmable bath  and equilibrated at 30°C for 10 min, before being heated at 0.25°C.minThe daily temperature conditions and thermal frequencies for four habitats, the coolest in the shade and the hottest in the sun, are shown for each shoreline in Figure E. malaccana snails. The overall magnitude of their LT50 adjustment was 2.9°C, when accounting for all laboratory-acclimated and field-acclimatized conditions , but no further increase in heat tolerance was observed for this population with further warming  significantly lowered the mean LT50 was greater in WS snails (57.7°C) compared to CS snails . When the cool and warm habitat data were combined (CS and WS), and compared with the data for WS snails, the combined data set showed significantly lower LT50 values for ECA, CA, and EWA  of Echinolittorina snails  acclimation between populations from thermally-different shorelines  and thermal thresholds  for different durations of thermal cycling . Whereas seasonal acclimatization, regulated by isozyme expression , is typiresholds . Previouulations , such thce versa . The samund 45°C . Interesund 45°C .Echinolittorina snails. Likewise, minimum field temperatures were largely invariable across the habitats and shores, which excludes these temperatures as potential determinants of the lower boundary for heat tolerance plasticity. The limit to this boundary, assessed during CA for both shores, possibly relates to the loss of heat-hardening, a mechanism which should be beneficial by eliminating costs associated with the upregulating and functioning of Hsps  were largely similar across habitats (sunned and shaded) and shorelines  is restricted to high-shore species or whether it is independent of vertical distribution on the shore. Because lower-shore habitats are strongly stabilized by the seawater temperature, and because the seawater temperature is largely invariable across nearby shorelines, the shoreline effect should be lessened in the lower-shore. In addition to suggesting that seawater temperature stability is likely to restrict thermal acclimation selection in lower-shore tropical species, we propose as a testable hypothesis that non-reversible plasticity should also be more constrained in lower-shore compared to higher-shore species.Acclimation capacity (the degree of change in a trait following cool or warm laboratory acclimation) is becoming a key measure of the vulnerability of ectothermic animals to future warming . An earlEmerging from this study is a second methodological issue relating to diel cycling of the laboratory acclimation temperature. This is not only important considering that such cycling occurs naturally, but also in terms of initiating an acclimatory HSR. If the primary heat-hardening response requires that exposure temperature surpasses an HSR induction threshold, then acclimation temperatures that are stable or fluctuate below this threshold will conceivably not yield an acclimatory response, leading to erroneous conclusions. This further highlights the need to determine the HSR threshold temperature prior to developing acclimation protocols in marine intertidal animal studies.Our study adds an important dimension to the existing theory proposing that thermal tolerance plasticity is relatively constrained in tropical ectotherms. In particular, it reveals that this plasticity can be complex in thermally-heterogeneous tropical marine intertidal ecosystems. Whereas the contribution of marine intertidal circumstances to a body of theory for thermal plasticity developed largely from terrestrial and subtidal animals might be questionable, our findings nonetheless caution against the indiscriminate use of this theory when interpreting intertidal data. We further show that without critical consideration of the thermal heterogeneity at the scale of the organism and how this heterogeneity may drive different forms of thermal tolerance plasticity, investigations risk generating misleading conclusions.AB and DM developed the hypothesis and prepared the manuscript. AB and NM carried out the experimental laboratory work. DM undertook the field recordings. NM reviewed 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.


The pulvinar influences communication between cortical areas. We use fMRI to characterize the functional organization of the human pulvinar and its coupling with cortex. The ventral pulvinar is sensitive to spatial position and moment-to-moment transitions in visual statistics, but also differentiates visual categories such as faces and scenes. The dorsal pulvinar is modulated by spatial attention and is sensitive to the temporal structure of visual input. Cortical areas are functionally coupled with discrete pulvinar regions. The spatial organization of this coupling reflects the functional specializations and anatomical distances between cortical areas. The ventral pulvinar is functionally coupled with occipital-temporal cortices. The dorsal pulvinar is functionally coupled with frontal, parietal, and cingulate cortices, including the attention, default mode, and human-specific tool networks. These differences mirror the principles governing cortical organization of dorsal and ventral cortical visual streams. These results provide a functional framework for how the pulvinar facilitates and regulates cortical processing. The pulvinar is involved in vision and attention, but its interactions with other brain regions are little-studied. Here, using fMRI the authors show that the human pulvinar has widespread functional coupling with cortical areas that reflects its intrinsic organization and the topographic layout of cortex. The thalamus is extensively interconnected with cortex, and, as a general principle, cortical areas that are directly connected to one another are also indirectly connected via the thalamus6. Thus, anatomically, the thalamus is well positioned to influence cortical function. Within the posterior thalamus, the pulvinar is thought to be critical for active vision by regulating visual cortical processing9. What are the organizing principles that enable the pulvinar, a relatively small region, to coordinate communication across the large, functionally diverse landscape of visual cortex?The primate cortex comprises a mosaic of functionally specialized regions. A major focus in neuroscience has been to understand how these areas coordinate information to support our perception of the environment. One prominent view is that the thalamus facilitates and regulates communication between cortical areas10, and there is only a beginning understanding in the human brain that the pulvinar appears to be similar to other primate species13. However, the human brain shows a great number of human-specific adaptations, and it is unclear how they are reflected in the functional organization of the pulvinar, a vastly understudied region of the human brain.The primate pulvinar is anatomically and functionally heterogeneous. Most of our understanding of the pulvinar comes from studies in non-human primates  coexist and may vary as a function of subdivision.A straightforward set of predictions can be derived from the pulvinar’s known anatomical connectivity in non-human primates. The pulvinar’s anatomical connections appear to be topographically organized such that neighboring parts of cortex project to neighboring parts of the pulvinarWhile much is known about the anatomical cortical connections in the pulvinar, little is known about the fine-grained functional coupling between the pulvinar and cortical areas. Here, we utilized fMRI in humans and a large variety of experimental tasks to characterize functional organizing principles of the pulvinar. We systematically investigated functional specialization of the human visual pulvinar and its relation to cortical organization. In addition to previously reported retinotopic coding and attentional modulation, we found novel evidence that the human pulvinar is involved in object vision and temporal integration. Clusters selective for visual categories, such as faces and scenes, were identified within the posterior ventral pulvinar, similar to the organization of inferotemporal (IT) cortex. Further, the pulvinar was sensitive to the temporal structure of visual input. Activity in the pulvinar was synchronized across subjects during movie viewing and, within the dorsal pulvinar, the degree of this synchronization was modulated by the temporal continuity of the movie. Functional connectivity analyses revealed the presence of two prominent pulvino-cortical networks. The dorsal pulvinar was functionally linked with frontal, parietal, and cingulate areas involved in attentional control, tool processing, and the default mode network. The ventral pulvinar was functionally linked with occipital and temporal areas involved in form, object, and scene recognition. The organization of cortical coupling within the pulvinar reflected both the distances between cortical areas and their functional specialization. Collectively, these results demonstrate that the dorsal and ventral pulvinar are major nodes in the often cortically-emphasized dorsal and ventral visual processing streams and illustrate that principles guiding the functional organization of cortex are present within the human pulvinar. These data highlight the fine-grained organization of pulvino-cortical interactions and greatly contribute to our understanding of the human thalamus by illustrating important constraints on the role(s) of the pulvinar in facilitating and regulating cortical processing.p < 0.05, FDR-corrected). The anatomical extent of these contralateral maps corresponded to the locations of two visual field maps within the ventral pulvinar described previously21. In contrast, the dorsal pulvinar responded weakly to visual stimulation and did not show a clear visual hemifield preference in individual subjects or in the group average contrast maps. To further quantify the visual responses within the pulvinar, regions of interest within the ventral and dorsal pulvinar were identified based on group average retinotopic maps and foci of functional connectivity with parietal and frontal cortex  respectively. The degree of contralateral tuning, as assessed by a d prime laterality index contrasting evoked activity from contralateral and ipsilateral visual stimulation . Responses in the dorsal and ventral pulvinar also were differentiated based on the degree of attentional modulation. The dorsal pulvinar showed greater attentional modulation than the ventral pulvinar, as assessed by a d prime attentional modulation index contrasting contralateral visual stimulation during covert attention from contralateral stimulation during central fixation (t(4) = 3.16, p = 0.034, n = 5). These results are consistent with prior literature showing contralateral tuning and attentional modulation in the pulvinar22. Together, these data illustrate a differentiation between the ventral and dorsal pulvinar based on visual responsiveness and attentional modulation, mirroring the broad distinctions of occipital and fronto-parietal cortices.Activity within the pulvinar varied as a function of stimulus location, attentional allocation, and context Fig. . The venion Fig. , right, 23 that measures the temporal similarity of activity evoked by an audiovisual movie across individuals. Responses in both the dorsal and ventral pulvinar during viewing of intact movies were consistent across individuals , demonstrating that the human pulvinar exhibits stereotyped activity during the processing of naturalistic stimuli. However, only the ventral pulvinar showed consistent responses across individuals for viewings of permuted versions of the same movie where the temporal structure and narrative flow were disrupted. Subtracting the response consistency of the scrambled movie from the intact movie yielded focal bilateral clusters of voxels within the dorsal pulvinar . These results mirror a previously established hierarchy of temporal receptive windows across occipital, parietal, and frontal cortices24. That is, activity in the ventral pulvinar reflects moment-to-moment transitions in visual statistics, similar to occipital visual areas, whereas activity in the dorsal pulvinar reflects attentional and contextual information, similar to frontal and parietal areas.The pulvinar was also sensitive to the temporal structure of visual stimulation. Sensitivity to temporal structure was assessed by an inter-subject correlation (ISC) approachp < 0.05, FDR-corrected, n = 16). Face selectivity was apparent at the individual subject level. Mean beta coefficients were extracted from this region in individual subjects using a leave-one-out analysis to avoid issues of circularity . Faces consistently evoked a larger response than scenes in individual subjects . The same region of the ventral pulvinar also showed a preference for faces vs. objects and faces + headless bodies vs. objects + scenes . Across all five categories, faces and headless bodies evoked the largest responses in the posterior pulvinar as compared with scenes, scrambled images, and objects . For most cortical areas, the mean timeseries of activity was positively correlated with the timeseries of many voxels in the pulvinar. In addition, most cortical areas were positively correlated with each other, making it difficult to evaluate the spatial specificity of the correlated signal. We assumed that signals between directly connected areas should be more correlated than between indirect or weakly connected areas. Therefore, we reasoned that assessing the spatial correlation pattern across all areas should be a more sensitive measure  for localizing cortical coupling within the pulvinar. To do this, we first calculated the mean timeseries of activity for each cortical area and computed temporal correlations between all cortical area pairs . We then computed the temporal correlation between the mean timeseries of each cortical area with the timeseries of each voxel in the pulvinar . For each subject, we then compared the pattern of the voxel-wise pulvino-cortical temporal correlations with the (leaving that subject out) pseudo-group average cortical area correlation profile, yielding a voxel-wise measurement  of the similarity between each cortical area’s correlation profile and every pulvinar voxel’s cortical area correlation profile. This measurement is referred to as the pulvino-cortical connectivity. A complete pulvino-cortical connectivity map within the pulvinar was generated for each cortical area in each subject and was used for all subsequent network-level analyses.The pulvinar was functionally coupled with visual cortex even in the absence of visual stimulation. We assessed the similarity of within-hemisphere activity between the pulvinar and 39 cortical areas across occipital, temporal, parietal, cingulate and frontal cortices using Pearson correlations. These areas tile visual and associated cortex and were chosen as they share broad functional similarity  yet vary in their specialization and cortical location. Correlations for a subset (23) of these areas were previously reportedn = 13) broadly reflected a functional distinction between dorsal and ventral visual streams . The other cluster comprised all parietal and frontal regions  as well as the retrosplenial cortex (RSC). For both clusters, most areas were linked with many other areas within the cluster  or no (r < = 0) links between clusters. The notable exception was RSC, which had several links with both clusters, indicating that it may serve as a bridge between dorsal and ventral clusters.Network-level analyses on the group average pulvino-cortical connectivity  and between averages from subsampling subjects . MDS and clustering on the group average of each hemisphere separately yielded similar structure  for neighboring cortical areas tended to have a good degree of overlap in the pulvinar and there was a linear relationship between the degree of overlap (Dice’s coefficient) and the cortical distance between seed areas  = −0.48, p < 0.0001). This relationship was driven by correlations within the occipito-temporal cluster (r(274) = −0.63, p < 0.0001) and was not apparent within the fronto-parietal cluster (r(103) = −0.10, p = 0.31). The difference between the two cluster correlation coefficients was significant . Taken together, these data demonstrate a broad distinction between the dorsal and ventral pulvinar’s cortical coupling and the functional organization within each subdivision.The pulvino-cortical connectivity maps  = 0.82, p < 0.0001). A second cluster (red) contained IPS5 and regions in anterior parietal (antPPC) and lateral temporal (latTemp) cortex that we, and others, have shown to form a human-specific tool network36. Within this cluster, there was no clear relation between the distances of pulvino-cortical connectivity peaks and cortical distance. The third cluster (blue) contained two regions in the IPL and the PreC that are associated with the default mode network37 as well as two additional regions in medial (medPPC) and lateral parietal cortex (latPPC) . Within this cluster, distances between peaks of pulvino-cortical connectivity reflected functional similarity, not cortical distance. Specifically, the pulvino-cortical connectivity peaks of the functionally similar inferior parietal and posterior cingulate/precuneus areas were within 1 mm of each other, and the other two lateral and medial parietal areas were within 0.5 mm of each other. The peak of latPPC (and medPPC) and the peak of IPL (and PreC) connectivity maps were both separated by several millimeters even though these areas are cortically adjacent. Further, the axes of organization within each cluster did not map across other clusters, suggesting that the organization of areas within a cluster does not translate to other clusters. For example, the anterior-posterior axis reflected cortical distance within the first cluster containing most IPS and frontal maps but did not for the other two clusters. RSC was not included in these analyses since it had several links with both pulvinar clusters . Functional coupling of face-selective areas  and scene-selective area PPA was localized to the ventral posterior pulvinar  and intermediate  visual cortex, which likely comprise the inputs. Clustering on this subset of areas yielded 4 clusters  = −0.72, p < 0.0001), suggesting that this was the prominent organizing factor of cortical coupling with the ventral pulvinar. Cortical distance relative to primary visual area V1 best accounted for the distribution of pulvino-cortical connectivity peaks within the ventral pulvinar  to temporal (AT) cortical areas. Clustering of pulvino-cortical connectivity within the ventral pulvinar revealed 2 clusters segmenting lateral and medial portions Fig. . This clers Fig. . One clu40 that are similar to the visual maps of the ventrolateral pulvinar and central lateral region of the inferior pulvinar in other primate species44. In contrast, the dorsal pulvinar was visually responsive to stimulation of both contralateral and ipsilateral visual space, but showed increased activity during directed spatial attention47 similar to fronto-parietal cortex; also see49. These results are consistent with lesion studies that have shown that damage to the pulvinar affects the allocation of attention, integration of visual information, and filtering of irrelevant information52 as well as with single unit recordings in monkeys that suggest that sub-regions of the pulvinar are involved in spatial attention53 and reflect awareness rather than solely the physical features of visual stimuli54. Our work exceeds previous investigations by identifying functional organizing principles of the human visual thalamus across a large variety of experimental tasks and in relation to cortical networks spanning occipital, temporal, parietal, and frontal lobes. Our report illustrates several novel findings that we will emphasize in the following discussion.The human pulvinar was engaged in a variety of visual tasks. The ventral pulvinar showed strong tuning for contralateral visual space, consistent with previous work demonstrating that the human pulvinar contains at least two retinotopic maps23. This suggets that the ventral pulvinar tracks moment-to-moment variations in low-level visual features. In contrast, the dorsal pulvinar was synchronized across subjects only for intact movie presentations, suggesting that it is generally insensitive to low-level stimulus features. Instead, this indicates that the dorsal pulvinar is involved in cognitive processes such as the tracking of narrative and real-life events that require the integration of information over several seconds and minutes55. This divergence in temporal sensitivity between the ventral and dorsal pulvinar parallels the hierarchy of temporal receptive windows spanning early visual cortex to higher-order parietal and temporal regions23. Together, these results demonstrate that the thalamus accumulates and processes information at multiple timescales.First, the pulvinar is sensitive to temporal structure. Activity in the pulvinar was synchronized across subjects during the viewing of a live-action movie. Interestingly, the temporal window of synchronization differed between ventral and dorsal sub-regions. The ventral pulvinar was synchronized across subjects even when frames of the movie were presented out of order, disrupting the narrative and temporal structure of movie events56 and projections from ventral temporal cortex (around area TF) to lateral portions of the pulvinar38. Similarly, an electrical stimulation study demonstrated evoked activity within the ventral medial pulvinar from stimulation of face-selective clusters in the lower bank of the STS57. Previous studies have demonstrated responsiveness in the monkey pulvinar to object features at the neuronal level59. Here, we demonstrate that this information is spatially clustered in the human pulvinar similar to the organization of ventral temporal cortex. To our knowledge, this is the first demonstration of such functional clustering within the human thalamus. This is particularly notable given the extensive overlap of cortical projections throughout the pulvinar38, which could have had the effect of blurring out such functional specificity. While emphasis is often placed on the pulvinar’s role in visual attention, our results illustrate a distinct role in object vision, and more broadly illustrate that ubiquitous principles of cortical organization  hold for the thalamus despite considerable differences in architecture .Second, the pulvinar encodes high-level visual form information. A posterior medial region of the ventral pulvinar responded preferentially to face (vs. scene) images and was functionally coupled with face-selective temporal areas at rest. A lateral region of the ventral pulvinar showed weak selectivity for scenes (vs. faces) and was functionally coupled with scene-selective temporal areas at rest. These data are consistent with anatomical connectivity studies in macaques that showed projections from the medial ventral pulvinar to the lower lip of the STS14. The dorsal pulvinar was functionally coupled with parietal, frontal, and cingulate cortices, consistent with anatomical studies in other primates62. Functional coupling between the pulvinar and dorsal attention network areas  appeared to be situated within lateral parts of the dorsal pulvinar and potentially corresponds to the border between the dorsal lateral nucleus and the lateral extent of the medial pulvinar, similar to other primates30. In contrast, the foci of functional coupling with the cingulate and IPL were situated entirely within the medial pulvinar, similar to other primates63. This differentiation in cortical coupling, as well as visual tuning, highlights a functional divide between the dorsal and ventral pulvinar similar to what has been observed anatomically in other primates31. More broadly, the differences between the dorsal and ventral pulvinar mirror the divergence of functions between the dorsal and ventral cortical visual pathways27, and illustrate that, similar to cortex, the pulvinar is functionally organized into two major processing streams.Third, the pulvinar was functionally coupled with visual cortex even in the absence of visual stimulation. During rest, the pulvinar was functionally coupled with 39 functionally dissociable areas across occipital, parietal, temporal, frontal, and cingulate cortex. The mean activity of individual cortical areas was correlated with activity in focal regions of the pulvinar. The foci of these correlations tended to overlap in the pulvinar for neighboring cortical areas. Individual areas were functionally coupled to either the dorsal or ventral pulvinar, but generally not to both. The ventral pulvinar was functionally coupled with occipital and temporal cortices, consistent with studies in other primate species showing that these regions are anatomically interconnected64. There was a strong linear relationship between the Euclidean distances of cortical correlation peaks within the pulvinar and cortical distances between seed areas. While some aspects of cortical coupling within the ventral pulvinar were consistent with the functional specializations of occipital and temporal cortex, overall, correlations were better accounted for by anatomical distance. For example, though the foci of correlated activity for two face-selective areas partially overlapped within the ventral pulvinar and were distinct from the foci of correlated activity for a scene-selective area, these data reflected broader differences between ventral-lateral and ventral-medial temporal cortex. The lack of such clustering within the ventral pulvinar solely based on functional specialization may appear at odds with our finding of category selective clusters within the ventral pulvinar, which suggests the presence of functional clustering. However, this likely reflects a difference of spatial scale . Local clustering of functionally similar neurons  reflects a minimization of cortical distance. Therefore, local functional clustering such as face-selectivity within the ventral pulvinar is not at odds with a broader organization of pulvino-cortical coupling based on cortical distance. This is further supported by our finding that the face-selective region in the ventral pulvinar was correlated with a face-selective cortical region (FFA) in temporal cortex. In contrast to the ventral pulvinar, correlations between the dorsal pulvinar and frontal-parietal cortex predominantly reflected functional specialization. Spatial clustering of frontal-parietal correlations within the pulvinar differentiated the dorsal attention network from the default mode and tool networks. Further, the foci of correlated activity for areas such as IPS2 and FEF largely overlapped in the dorsal pulvinar despite their large cortical distances. At a finer scale, cortical distance was apparent in the spatial organization of correlations within the dorsal pulvinar for a subset of frontal-parietal cortical areas. The peaks of correlated activity for dorsal attention network areas were roughly distributed along an anterior-posterior axis within the dorsal pulvinar similar to their relative cortical distances. However, this relationship was not observed for the peaks of correlated activity of other cortical areas within the dorsal pulvinar. Altogether, these results show that the structure of cortical interactions varies throughout the pulvinar and suggest a fundamental difference in how the pulvinar interacts with dorsal and ventral cortical pathways.Fourth, the spatial organization of pulvino-cortical coupling reflected cortical distance and functional specialization. Within the ventral pulvinar, cortical correlations predominantly reflected cortical distance. The foci of correlated activity with occipital and temporal areas were distributed along a rostrolateral to caudomedial axis of the ventral pulvinar, similar to the distribution of cortical anatomical connections in the macaque pulvinar65, the human pulvinar maintains an organization of nuclei similar to other primate species65. Together, these data suggest that the expansion of individual nuclei allows for species-specific functional adaptations within the pulvinar while preserving its global organization across primates.The pulvinar incorporates human-specific adaptations. The dorsal medial pulvinar was functionally coupled with anterior parietal (antPPC) and lateral temporal (latTemp) cortical regions involved in the processing of tools. The foci of these correlations were located near the foci of other areas proximal to anterior parietal cortex, but these two areas  were grouped into a separate cluster, suggesting that the dorsal pulvinar has expanded to allow for interactions with human-specific cortical networks. Despite a substantial increase in size across primates9. Our results illustrate the fine-grained organization of pulvino-cortical interactions and highlight important constraints on these interactions. We propose that the dorsal pulvinar predominantly facilitates communication between widespread cortical areas that mediate top-down processes such as control of attention, as well as between nodes in the default mode and temporal-parietal tool networks. The ventral pulvinar predominantly facilitates communication between neighboring cortical areas involved in visual recognition and feature extraction, possibly via local competition66. Interactions between the dorsal and ventral pulvinar were not prominent in our data with the exception of retrosplenial cortex (RSC), which appeared to be a hub linking the two subdivisions. Additional interactions likely exist and may be mediated by a tertiary area such as the thalamic reticular nucleus (TRN). While not explored in the present study, the monkey pulvinar also contains extensive anatomical connections with other subcortical regions including the superior colliculus67 and the amygdala68. Future work will be needed to resolve how this organization relates to the organization of pulvino-cortical coupling. More broadly, it will be interesting to see whether cortical coupling with the anterior pulvinar and thalamic regions involved in non-visual sensory functions70 exhibit similar principles of organization. Lastly, the relationship between the functional organization of the pulvinar and individual pulvinar nuclei remains to be resolved. Though the localization of functional coupling for individual cortical areas within the pulvinar provides insight into such structure-function relationships, development of high-resolution anatomical MRI techniques that enable the precise delineation of nuclei is needed. Such data would facilitate further comparisons across primate species. Overall, our results demonstrate that the human pulvinar supports a diverse assortment of visual functions, mirroring those of visual cortex, and provide a functional framework for how the pulvinar facilitates and regulates cortical processing.The pulvinar has been shown to regulate communication within and between cortical visual areas11. In addition, all subjects participated in three experiments to localize cortical visual areas: (i) polar angle cortical mapping; (ii) eccentricity cortical mapping; (iii) memory-guided saccade mapping.Twenty-eight subjects  participated in the study, which was approved by the Institutional Review Board of Princeton University. All subjects were in good health with no history of psychiatric or neurological disorders and gave their informed written consent. Subjects had normal or corrected-to-normal visual acuity. Thirteen subjects (S1-S13) participated in task-free resting-state. Sixteen subjects  participated in the object localizer experiment. Eleven subjects  participated in the movie viewing experiment. Five subjects  participated in the laterality and attentional modulation experiments. To localize subcortical visual field maps, nine subjects  participated in a polar angle mapping session and five subjects  participated in an eccentricity mapping session using scanning protocols optimized for subcortical structuresfor more details, see72. Stimuli were projected from either a Christie LX650 liquid crystal display projector  or a Hyperion PST-100984 digital light processing projector (Psychology Software Tools) onto a translucent screen located at the end of the Siemens 3 T Allegra and Skyra scanner bores, respectively. Subjects viewed the screen at a total path length of ~60 cm through a mirror attached to the head coil. The screen subtended either 30°x 30° (Allegra), or 51°x 30° (Skyra) of visual angle. A trigger pulse from the scanner synchronized the onset of stimulus presentation to the beginning of the image acquisition.The stimuli were generated on Macintosh G4 and G5 computers (Apple) using MATLAB software (MathWorks) and Psychophysics Toolbox functions11. During the fixation scans, subjects were instructed to maintain fixation on a centrally presented dot (0.3° diameter) overlaid on a mean grey luminance screen background for 10 min. During the eyes closed scans, the projector was turned off and subjects were instructed to keep their eyes closed for 10 min. Two runs were collected per resting state condition.Thirteen subjects participated in two versions of resting state scans: (1) fixation; and (2) eyes closed74. Eleven subjects viewed an audiovisual movie clip from the 1975 commercial film Dog Day Afternoon75. Subjects were instructed to attend to the movie and to freely view it. Movie stimuli subtended 20° horizontally and 16° vertically. A 5 min. 45 s clip of the film was presented as well as a temporally scrambled version of the stimulus where the clip was broken into segments spanning 0.5–1.6 s and reordered. Each movie was presented twice.A timescale localizer was used to delineate regions of the pulvinar that were sensitive to short- and long- timescales following established procedures° horizontally and vertically. Subjects were instructed either to attend to a central fixation point (0.5°) and to detect changes in its luminance, or to maintain central fixation while covertly attending to one of the hemifields and to detect changes in luminance within a (varying) focal region of the checkerboard. Stimulus conditions were presented in 16 s blocks and were interleaved with 16 s periods where subjects maintained central fixation on a mean grey screen. The subjects’ task was varied between blocks. Each stimulus block was presented twice per run. Twelve runs were collected per scan session.Five subjects participated in an experiment designed to measure contralateral tuning and attentional modulation. Flickering checkerboard stimuli were presented to either the right or left visual hemifield. Stimuli subtended 15Retinotopic areas V1, V2, V3, V3A-B, hV4, VO1-2, PHC1-2, LO1-2, TO1-2, IPS0-5, SPL1, IFS, and FEF were identified using standard criteria (Supplementary Methods).Face-selective domains , body-selective domains , scene-selective domains , and the object-selective domain LOC were identified using standard criteria (Supplementary Methods). Due to copyright restrictions, the original images used in this localizer cannot be published. In Fig. 37. The lateral temporal (latTemp) and anterior parietal (antPPC) areas were identified based on relative anatomical location and MNI coordinates from our previous study on the tool network34. Though these tool-selective regions are often left hemisphere lateralized, we included bilateral ROIs because weak tool-selectivity was observed in homotopic locations within the right hemisphere34 and these regions are functionally coupled during rest76. Lateral (latPPC) and medial parietal (medPPC) areas were identified as regions located along the cortical surface in-between the IPS2-4 and the IPL and PreC, respectively. MNI coordinates were projected onto each subject’s native space anatomical surface. An ROI spanning 6 mm on the surface around each MNI- or anatomically-defined region was drawn for each area. Areas of overlap with surrounding retinotopic maps were excluded. For overlap between latPPC and IPL (as well as between medPPC and PreC), the border was adjusted to the midpoint. ROI size did not greatly affect analyses and results were qualitatively comparable for larger (8 mm) and smaller (4 mm) ROI sizes.The posterior cingulate/precuneus (PreC) and inferior parietal (IPL) areas were identified based on relative anatomical location and MNI coordinates from prior studies on the default mode network32 in MNI space was aligned to each subject’s native anatomical image using non-linear anatomical registration procedures outlined below. These 180 areas were used in a secondary analysis to evaluate pulvinar functional connectivity patterns based of correlations across the whole cortical surface.An atlas of 180 areas across the cortical surface of each hemisphere2 . For all other experiments, data were acquired with a Siemens 3 T Skyra scanner using 20-channel phased-array head(16)/neck(4) coil . All functional acquisitions used a gradient echo, echo planar sequence with a 64-square matrix  leading to an in-plane resolution of 3 × 3 mm2 . High-resolution structural scans were acquired in each scan session for registration to surface anatomical images .Functional MR images were acquired with a gradient echo, echo planar imaging (EPI) sequence using an interleaved acquisition. The specific parameters for each scan session are outlined below. For the laterality / attentional modulation scans, data were acquired with a Siemens 3 T Allegra scanner using a circularly polarized head coil . For laterality and attentional modulation experiments, 25 oblique slices were acquired using an EPI sequence with a 128-square matrix  leading to an in-plane resolution of 1.5 × 1.5 mm77, SUMA78, FSL FSL, RRID:birnlex_2067;79http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/81, FreeSurfer FreeSurfer, RRID:nif-0000-00304;83http://surfer.nmr.mgh.harvard.edu/, and MATLAB . Functional data were slice-time and motion corrected. Motion distance (estimated by AFNI’s 3dvolreg) did not exceed 1.0 mm (relative to starting head position) in any of the 6 motion parameter estimates (3 translation and 3 rotation) during any run for any subject.The data were analyzed using AFNI Analysis of Functional NeuroImages, RRID:nif-0000-00259;d’), defined by the following formula (1).μcontralateral and μipsilateral are the average responses to contralateral visual stimuli ; σcontralateral and σipsilateral are the SDs. Attentional modulation was calculated using the same formula between covert hemifield attention and central fixation conditions.Data were spatially filtered with a 4 mm (FWHM) Gaussian kernel, which increased signal-to-noise ratio (SNR) while maintaining good anatomical localization of signals within the pulvinar Fig. . A multiA leave-one-out analysis was performed to evaluate the profile of responses across visual categories within the pulvinar. For each subject, an n-1 pseudo-group average face-minus-scene contrast map was computed using a mixed effects meta-analysis (AFNI’s 3dMEMA) to identify a face-selective region of interest within the posterior medial pulvinar. The mean betas for each category were calculated within this region from the held-out subject’s data.85, though our results were not dependent on these preprocessing steps, and correlation analyses on the raw data yielded qualitatively and statistically similar results. Global mean signal (GMS) removal was not included in the analysis reported here given concerns about negative correlations88, though inclusion of GMS removal yielded statistically similar results. To minimize the effect of any evoked response due to the scanner onset, the initial 21.6 s were removed from each rest scan. To extract the mean signal from each cortical area, voxels that fell between the gray and white matter boundaries were mapped to surface model units (nodes). The mean signal from each cortical area was extracted into MATLAB for correlation analyses. Data within the thalamus was then spatially filtered with a 4 mm (FWHM) Gaussian kernel, which increased SNR and improved correspondence of correlation patterns between subjects while maintaining good anatomical localization of signals within the pulvinar.Several additional steps were performed on the data: (1) removal of signal deviation > 2.5 SDs from the mean (AFNI’s 3dDespike); (2) temporal filtering retaining frequencies in the 0.01-0.1 Hz band; (3) linear and quadratic detrending; and (4) removal by linear regression of several sources of variance: (i) the six motion parameter estimates (3 translation and 3 rotation) and their temporal derivatives, (ii) the signal from a ventricular region, and (iii) the signal from a white matter region. To avoid partial volume effects with surrounding grey matter, ventricular and white matter regions were identified by hand on each subject’s mean EPI image. These are standard preprocessing steps for resting-state correlation analyses73. This analysis provides a measure of the consistency of a response to the temporally complex stimulus  by comparing the BOLD response across different subjects. ISC helps avoid the contribution of idiosyncratic responses to correlation patterns and circumvents the need to specify a model for the neuronal processes in any given brain region during movie watching. For ISC, repetitions for each condition were averaged within subject. Data were then transformed to MNI space and voxel-wise correlations were computed between the responses in each subject with the average of all other subjects for each condition separately. This yielded a voxel-wise measure of the consistency of brain activity evoked by the audiovisual movie clip. Group average correlation maps were calculated, and the difference was computed between intact and scrambled correlation maps.For the movie viewing experiment, an inter-subject correlation (ISC) analysis approach was usedWe applied a two-step Pearson correlation analysis to identify cortical areas whose spatial profile of correlations with all other cortical areas  was similar to the cortical area correlation profile of individual voxels in the pulvinar. First, temporal correlations were performed between each cortical area and between cortical areas and the pulvinar. To identify the cortical correlation profile of individual cortical areas, the mean timeseries of each cortical area was correlated with the mean timeseries of every other cortical area in each subject. To identify the cortical correlation profile of the pulvinar in each subject, the mean timeseries of each cortical area was correlated with the timeseries of each voxel within a pulvinar mask.11, the computation of each pulvino-cortical connectivity map differs in the current study due to the inclusion of several (16) additional parietal, temporal, and frontal areas, which changes the size of the cortical connectivity profile. Though the cortical connectivity profile has been greatly expanded in this study, the resulting pulvino-cortical connectivity maps for the 5 individual cortical areas previously reported  were very similar to maps from the previous analyses, demonstrating the robustness of these connectivity measures.Next, we compared the pattern of cortical correlations (profiles) for individual pulvinar voxels with those of individual cortical areas. For each subject, the cortical correlation profile of each pulvinar voxel was correlated to the pseudo-group average cortical correlation profile of each cortical area, where the average excluded that subject. This yielded a measurement of similarity between each cortical area’s cortical correlation profile and every pulvinar voxel’s cortical correlation profile, which we refer to as the pulvino-cortical connectivity. Significant positive similarity in the pulvino-cortical functional connectivity was taken as evidence for connectivity between a pulvinar voxel and a given cortical area. Individual subject anatomical volumes were then aligned using a two-step linear (AFNI’s 3dAllineate), nonlinear (AFNI’s 3dQwarp) registration procedure, and each subject’s pulvino-cortical functional connectivity profile was transformed into MNI space. Voxel-wise, one sample t-tests were used to assess statistical significance. Even for the 23 cortical areas that were investigated previously89. The correspondence between the EPI acquisition grid and anatomy naturally varies across individuals; thereby leading to variability in the degree to which individual voxels sample functionally distinct regions in the pulvinar across individuals. Averaging voxels across subjects reduces effects in voxels where the signals are less prevalent across subjects, thereby increasing the spatial precision. The degree to which this averaging improves spatial precision is limited by the accuracy of anatomical registration across individuals. However, nonlinear registration algorithms can handle the alignment of subcortical structures across individuals given the uniformity of subcortical structures . e.g., previously, we showed that pulvino-cortical connectivity maps with V1 overlapped with two prominent retinotopic maps within the ventral lateral pulvinar whereas connectivity maps of motion-sensitive region TO overlapped with a medial portion of the ventral pulvinar. This spatial precision was clear in the group average while the distinction between these maps was less clear in individual subjects.While functional connectivity profiles for individual subjects are limited in spatial precision by the imaging acquisition resolution, group average maps can yield a finer spatial precision than the original sampling resolution as has been previously demonstrated in group average subcortical imaging32.As a control analysis to ensure that the connectivity patterns for the 39 areas used in the main analysis were not dependent on the particular ROIs, areal connectivity profiles were calculated using an 180 ROI parcellation of the whole cortical surface2|A ∩ B| / (|A| + |B|). The group average map for each cortical area was threshold at p < 0.05 and binarized. Degree of overlap was assessed relative to the entire anatomical volume of the pulvinar.To compare the spatial localization of correlation patterns within the pulvinar, the overlap of pulvino-cortical connectivity maps was computed for all areas pairs with Dice’s coefficient, To assess the structure of the pulvino-cortical connectivity, correlations were calculated between the pulvino-cortical connectivity maps of each cortical area. This high-dimension 39 × 39 connectivity matrix was converted to a dissimilarity (Euclidean distance) matrix and classical multi-dimensional scaling (MDS) was applied. The first two principal dimensions were visualized Fig. . To comp28 from the Brain Connectivity Toolbox29. This algorithm automatically subdivides a (weighted) network into non-overlapping groups that maximizes the number of within-group links and minimizes the number of between group links. The algorithm starts with one group and continues to split the data into subgroups until additional splits yield no further minimization of between-group edges (as assessed by whether the signs of the eigenvectors are uniform). That is, the algorithm determines the optimal cluster size, which is an advantage over other clustering approaches, such as k-means. In practice, we found that k-means with a k matched to the cluster size returned from this spectral clustering approach yielded qualitatively similar divisions that would not change interpretation of our results.Clustering on the group average data was performed using a spectral (eigendecomposition) algorithmEuclidean distances between the group average centroids for all 39 areas were calculated using AFNI’s SurfDist. Cortical distance measures were correlated with Dice’s coefficient (Sorensen index) and with the Euclidean distances between peaks in the pulvino-cortical connectivity maps.p < 0.05. MDS and clustering analyses were performed on the individual and group-average data. Pearson correlations were performed between the spatial pattern of correlations and cortical distance. Eleven subjects participated in a movie viewing experiment. Patterns of co-fluctuations in the BOLD signal were assessed between subjects using a Pearson’s correlation. Five subjects participated in the contralateral tuning and attentional modulation experiment. Response magnitudes and significance were estimated for each subject using a regression analysis. Group-level statistical significance was assessed across subjects using a one-sample, two-tailed t-tests. The data were corrected for multiple comparisons (FDR) at p < 0.05. A d prime index was used to calculate degree of contralateral tuning and attentional modulation across subjects. Statistical significance was assessed across subjects using a two-sample, two-tailed t tests. Sixteen subjects participated in the object localizer experiment. Voxel-wise response magnitudes and significance were estimated for each subject using a regression analysis. Voxel-wise group-level statistical significance was assessed across subjects using a mixed effects meta-analysis that models both within- and across- subject variability. For the ROI analysis, statistical significance was assessed across subjects using non-parametric testing (Wilcoxon signed rank test) and effect sizes (Cohen’s d) were calculated.Thirteen subjects participated in resting state scans. Individual and group-level analyses were performed. Patterns of co-fluctuations in the BOLD signal were assessed within subject using Pearson correlation. Group-level statistical significance was assessed for the spatial pattern of pulvinar correlations by Fisher transforming individual subject correlations and performing one-sample, two-tailed t-tests. Data were corrected for multiple comparisons (FDR) at Peer Review FileSupplementary Information


Extracellular vesicles (EVs) are naturally occurring cell-secreted nanoparticles that play important roles in many physiological and pathological processes. EVs enable intercellular communication by serving as delivery vehicles for a wide range of endogenous cargo molecules, such as RNAs, proteins, carbohydrates, and lipids. EVs have also been found to display tissue tropism mediated by surface molecules, such as integrins and glycans, making them promising for drug delivery applications. Various methods can be used to load therapeutic agents into EVs, and additional modification strategies have been employed to prolong circulation and improve targeting. This review gives an overview of EV-based drug delivery strategies in cancer therapy. The term extracellular vesicle (EV) refers to a wide variety of biological nanoparticles that engage in cell-to-cell communication (paracrine and autocrine) within micro and macroenvironments Despite substantial contributions to cancer treatment, chemotherapy is prone to rapid clearance, poor bioavailability, low intratumoral delivery, unspecific cytotoxicity, and consequent systemic side effects, frequently followed by the onset of tumor resistance The ability to obtain EV populations that display favorable properties could enable exploitation of complex multifaceted drug delivery mechanisms while circumventing complicated manufacturing processes. However, one of the major limitations of EV-based drug delivery has been the lack of efficient isolation methods. Although a variety of options exist for concentrating EVs from biological sources, including centrifugation  Figure A, size eNano-sized particles are recognized by the immune system through various processes, many of which originate from defense mechanisms against bacteria and viruses, which have similar dimensions. Immunorecognition often leads to nanoparticle accumulation in the liver due to size-dependent infiltration through vascular fenestrations An important aspect of using EVs for cancer therapy is intratumoral accumulation. A commonly proposed mechanism for nanoparticle deposition in tumors is the enhanced permeability and retention (EPR) effect, which was first described in 1986 Conventional chemotherapy does not exploit the EPR effect, as small molecules distribute easily in tissues in the absence of vascular fenestrations. In the case of EVs, that have short circulation times, the EPR effect is less likely to be a determining factor for tumor accumulation. However, long circulation times are not a necessity for tumor targeting, as there are other mechanisms that can enhance site-specific delivery. For example, if EVs display prominent preferential interactions with tumor vasculature, a few passes through the circulation could be adequate to achieve high intratumoral accumulation. It is also worth noting that the final localization of EVs in the body may not necessarily reflect that of the therapeutic cargo. It was recently demonstrated that EVs and various RNA cargo display different biodistribution patterns, suggesting that cargo re-packaging and further transportation takes place in the body in vitro, there are several safety concerns with the use of these strategies in vivoEVs can be loaded with various therapeutic agents, including chemotherapy and nucleic acids, such as messenger RNAs (mRNA), miRNAs, small interfering RNAs (siRNAs), and small nucleolar RNAs (snoRNAs) Pre-loading techniques involve incorporating cargo into cells that encapsulate the material during EV production. Cells can package both biologically produced components (proteins and nucleic acids) and synthetic compounds. For example, treating cancer cells with chemotherapeutic agents leads to the production of drug-containing EVs Furthermore, genetic engineering of cells promotes endogenous packaging of overexpressed biomolecules A simple strategy for loading therapeutic agents into EVs is mixing with free drugs. This approach is more efficient for hydrophobic compounds compared to hydrophilic ones Electroporation involves the use of an electric field to induce spontaneous pore formation in lipid membranes Figure A. The prSaponin is a detergent that selectively removes membrane cholesterol, opening pores in lipid membranes Figure F 72. SapPrior to the discovery that EVs play a role in intercellular communication, they were thought to be solely involved in the elimination of waste products from cells Altering the surface of EVs can impact biodistribution and targeting capabilities in vivo silencing of RNAs in brain regions of mice was determined following intravenous administration of targeted and non-targeted EVs EV membranes have endogenous proteins that can be fused with targeting ligands through cell engineering. This technique was first reported in 2011 in a study where dendritic cells were transfected with plasmids encoding a fusion protein for lysosome-associated membrane protein 2 (Lamp2) and the central nervous system-specific peptide rabies viral glycoprotein (RVG) A similar fusion protein-based EV-engineering strategy was developed for treatment of chronic myelogenous leukemia (CML). Although the five-year survival of CML can be drastically improved with conventional therapy, i.e. tyrosine kinase inhibitors (TKIs), a subset of patients develop drug resistance and/or suffer from adverse side effects due to inefficient site-specific accumulation In vitro analysis showed that iRGD-EVs were taken up faster and to a greater extent in MDA-MB-231 breast cancer cell lines (expressing ITGαv), compared to untargeted dendritic cell EVs. Confocal microscopy demonstrated that iRGD-EVs co-localized with cancer cell membranes in a mere five minutes, while non-targeted EVs took 60 minutes to display a similar degree of co-localization. Flow cytometry demonstrated uptake efficiencies of 95.4% and 35.0% after two hours for iRGD-EVs and control EVs, respectively in vivo study, reduced cardiotoxicity was observed with targeted EVs compared to non-targeted In another study, dendritic cells overexpressing Lamp2b fused to the internalizing arginine-glycine-aspartic acid (iRGD) peptide targeting αv ITG were used to generate EVs In addition to utilizing EV membrane proteins, studies have indicated that fusion proteins incorporating hydrophobic transmembrane ligands can serve to enrich targeting ligands on the cell surface 4 construct. The resulting EVs were loaded passively through incubation with streptavidin -bound horseradish peroxidase (HRP). The EVs were able to deliver active streptavidin-HRP into B16F10 cells with six times higher uptake levels compared to incubation with the free protein Other EV-based engineering strategies for incorporation of targeting ligands include the use of post-isolation click chemistry, such as copper-catalyzed azide-alkyne cycloaddition. Click chemistry is a method by which ligands can be added to the surface of EVs without the use of solutions that damage biological components Overall, incorporation of targeting ligands in EVs has demonstrated promising results in increasing delivery of therapeutic agents into specific cells and organs, some of which are shielded by specialized obstacles, such as the blood brain barrier. However, as targeted synthetic nanoparticles have consistently failed in the clinic + and Cl- into the endosome contributing to osmotic swelling and rupture in vitro studies have demonstrated enhanced drug release from DEAP-EVs at pH 6.5 compared to pH 7.4 Aside from adding targeting ligands to the EV membrane surface, functionality can be introduced by adding peptides that are sensitive to the environment. Tumors have an acidic extracellular microenvironment (pH 6.5 -7.2), while all cells have an acidic intracellular endosomal environment (pH 5.0-6.5) compared to the physiological pH of 7.4 Another mechanism for pH-sensitive intracellular drug release was demonstrated using EVs incorporating a pH-sensitive glutamic acid-alanine-leucine-alanine (GALA) peptide Aside from adding targeting ligands to the surface of EVs, transport properties can be changed be removing endogenous surface molecules. The protein and RNA composition of EVs has been extensively studied, while the perhaps equally important glycome has been overlooked In the context of synthetic nanoparticles, various strategies have been developed to avoid activation of the immune system, including polymer coatings that reduce interactions with cells Many cell types have been used as sources for EV-based drug delivery, including mesenchymal stem cells (MSCs), immune cells, and cancer cells in vitro in response to specific growth factors MSCs are multipotent stem cells mainly found in bone marrow and adipose tissue. These cells are capable of differentiating into osteoblasts, chondrocytes, and adipocytes EVs secreted from immune cells are particularly promising for cancer therapy. For example, one of the mechanisms by which natural killer (NK) cells cause cancer cell death is through secretion of EVs containing cytotoxic proteins, such as perforin (PFN), granzyme A (GzmA), GzmB, granulysin (GNLY) and Fas ligand (FasL) Cancer cells produce a large amount of EVs with unique homing abilities, due to microenvironmental conditions characterized by rapid metabolism and hypoxia that drive metastatic processes ex vivo exposure to melanoma cells, while splenocytes from mice injected with CpG or a mixture of CpG and EVs did this to a much lesser extent Ex vivo exposure of these splenocytes to a lymphocyte cell line did not result in significant IFN-γ secretion or CTL activation, indicating melanoma cell line-specific immune responses. Additionally, cancer cell line-specific humoral responses were also observed in response to CpG-EV administration in mice. CpG-EVs were capable of delaying tumor growth, reducing metastasis, and prolonging survival when administered prior to or after cancer cell inoculation, while treatment with a mixture of CpG and EVs led to negligible antitumor effects One of the main advantages of using cancer cell-derived EVs for therapeutic purposes is the presence of tumor-specific antigens that can prime immune cells to induce an immune response. Loading EVs with inhibitors of immunosuppressive cells or immunostimulatory compounds can further enhance therapeutic efficacy. For example, murine melanoma cells transfected with a plasmid encoding a fusion protein composed of streptavidin and the EV-enriched protein, lactadherin, enabled subsequent surface tethering with a cytosine-phosphate-guanine (CpG) DNA adjuvant linked to biotin, a natural streptavidin ligand EVs may infer advantages over synthetic nanoparticles due to intrinsic targeting capabilities, complex surface proteins, and immunoevasive properties. However, there are advantages and challenges to both modes of drug delivery. Compared to EVs, synthetic nanoparticles can be produced in a more consistent manner resulting in a homogenous population. Additionally, first and second-generation synthetic nanoparticles, some of which entered the market as early as 1995 It is important to note that EV-based drug delivery studies require further side-by-side comparisons with synthetic nanoparticle controls in order to justify the use of the former. Liposomes with similar features as EV-based delivery systems  should be used for comparison purposes in vitro studies have demonstrated cancer-targeting capabilities of RBC-nanoparticles following modification with a targeting ligand Another drug delivery system that exploits natural membranes is cell membrane-coated nanoparticles (CMNPs). Comparisons with EVs should be discussed, as these carriers display similar size and surface properties. Similarly to EVs, the performance of CMNPs is highly dependent on the biological source material. For example, studies comparing erythrocyte, leukocyte, and platelet membrane coatings have noted differences in circulation time and tissue targeting CMNPs are being explored as alternatives to EV-based drug delivery systems due to concerns about complex and inefficient isolation methods resulting in low yields EVs offer promising prospects for improving the delivery of therapeutic agents due to intrinsic properties, such as tissue tropism. EVs have also been further engineered to maximize drug delivery potential, including incorporation of targeting ligands, stimuli-responsive components, and immunoevasive factors. Implementation of engineering strategies that have taken decades to optimize in the field of synthetic cancer nanomedicine could provide many advantages, resulting in drug delivery systems that benefit from both biological and exogenous properties.Exploiting several endogenous EV surface molecules could potentially be a superior strategyfor achieving organotropic targeting. However, this type of approach necessitates isolation of distinct EV subpopulations that display favorable transport properties, which has proved challenging with current techniques. Furthermore, EV-based transport in the body is not well understood, partially due to a lack of studies exploring the EV-protein corona interface.In addition to limitations in separating EV subpopulations and understanding EV transport properties, scalable manufacturing remains a major hurdle for clinical translation. It is also unclear whether allogeneic EV sources, which have the potential to be streamlined and developed as off-the-shelf products, could be used for future drug delivery applications. MSC-EVs are considered particularly suitable for drug delivery applications, as they have few immunostimulatory surface markers. However, studies evaluating the allogeneic potential of EVs from various sources are lacking. Plasma transfusions rarely result in adverse immune reactions The first clinical trials utilizing EVs were reported in 2005