eLife Assessment

This valuable work describes results from a set of simulation and empirical studies of a set-up assessing exploratory behavior in a potentially rewarding environment that contains danger. The core idea is that an instrumental agent can be helped to be both effective and safe, thus avoiding excessive danger, during exploratory behavior, if its influence is flexibly gated by an independent Pavlovian fear learning system. The conclusion that safe, but effective exploration can be achieved based on a flexibly weighted combination of a Pavlovian and an instrumental agent is solid.

Reviewer #1 (Public review):

Summary:

This paper provides a computational model of a synthetic task in which an agent needs to find a trajectory to a rewarding goal in a 2D-grid world, in which certain grid blocks incur a punishment. In a completely unrelated setup without explicit rewards, they then provide a model that explains data from an approach-avoidance experiment in which an agent needs to decide whether to approach or withdraw from, a jellyfish, in order to avoid a pain stimulus, with no explicit rewards. Both models include components that are labelled as Pavlovian; hence the authors argue that their data show that the brain uses a Pavlovian fear system in complex navigational and approach-avoid decisions.

In the first setup, they simulate a model in which a component they label as Pavlovian learns about punishment in each grid block, whereas a Q-learner learns about the optimal path to the goal, using a scalar loss function for rewards and punishments. Pavlovian and Q-learning components are then weighed at each step to produce an action. Unsurprisingly, the authors find that including the Pavlovian component in the model reduces the cumulative punishment incurred, and this increases as the weight of the Pavlovian system increases. The paper does not explore to what extent increasing the punishment loss (while keeping reward loss constant) would lead to the same outcomes with a simpler model architecture, so any claim that the Pavlovian component is required for such a result is not justified by the modelling.

In the second setup, an agent learns about punishments alone. "Pavlovian biases" have previously been demonstrated in this task (i.e. an overavoidance when the correct decision is to approach). The authors explore several models (all of which are dissimilar to the ones used in the first setup) to account for the Pavlovian biases.

Strengths:

Overall, the modelling exercises are interesting and relevant and incrementally expand the space of existing models.

Weaknesses:

I find the conclusions misleading, as they are not supported by the data.

First, the similarity between the models used in the two setups appears to be more semantic than computational or biological. So it is unclear to me how the results can be integrated.

Secondly, the authors do not show "a computational advantage to maintaining a specific fear memory during exploratory decision-making" (as they claim in the abstract). Making such a claim would require showing an advantage in the first place. For the first setup, the simulation results will likely be replicated by a simple Q-learning model when scaling up the loss incurred for punishments, in which case the more complex model architecture would not confer an advantage. The second setup, in contrast, is so excessively artificial that even if a particular model conferred an advantage here, this is highly unlikely to translate into any real-world advantage for a biological agent. The experimental setup was developed to demonstrate the existence of Pavlovian biases, but it is not designed to conclusively investigate how they come about. In a nutshell, who in their right mind would touch a stinging jellyfish 88 times in a short period of time, as the subjects do on average in this task? Furthermore, in which real-life environment does withdrawal from a jellyfish lead to a sting, as in this task?

Crucially, simplistic models such as the present ones can easily solve specifically designed lab tasks with low dimensionality but they will fail in higher-dimensional settings. Biological behaviour in the face of threat is utterly complex and goes far beyond simplistic fight-flight-freeze distinctions (Evans et al., 2019). It would take a leap of faith to assume that human decision-making can be broken down into oversimplified sub-tasks of this sort (and if that were the case, this would require a meta-controller arbitrating the systems for all the sub-tasks, and this meta-controller would then struggle with the dimensionality j).

On the face of it, the VR task provides higher "ecological validity" than previous screen-based tasks. However, in fact, it is only the visual stimulation that differs from a standard screen-based task, whereas the action space is exactly the same. As such, the benefit of VR does not become apparent, and its full potential is foregone.

If the authors are convinced that their model can - then data from naturalistic approach-avoidance VR tasks is publicly available, e.g. (Sporrer et al., 2023), so this should be rather easy to prove or disprove. In summary, I am doubtful that the models have any relevance for real-life human decision-making.

Finally, the authors seem to make much broader claims that their models can solve safety-efficiency dilemmas. However, a combination of a Pavlovian bias and an instrumental learner (study 1) via a fixed linear weighting does not seem to be "safe" in any strict sense. This will lead to the agent making decisions leading to death when the promised reward is large enough (outside perhaps a very specific region of the parameter space). Would it not be more helpful to prune the decision tree according to a fixed threshold (Huys et al., 2012)? So, in a way, the model is useful for avoiding cumulatively excessive pain but not instantaneous destruction. As such, it is not clear what real-life situation is modelled here.

A final caveat regarding Study 1 is the use of a PH associability term as a surrogate for uncertainty. The authors argue that this term provides a good fit to fear-conditioned SCR but that is only true in comparison to simpler RW-type models. Literature using a broader model space suggests that a formal account of uncertainty could fit this conditioned response even better (Tzovara et al., 2018).

Reviewer #2 (Public review):

Summary:

The authors tested the efficiency of a model combining Pavlovian fear valuation and instrumental valuation. This model is amenable to many behavioral decision and learning setups - some of which have been or will be designed to test differences in patients with mental disorders (e.g., anxiety disorder, OCD, etc.).

Strengths:

(1) Simplicity of the model which can at the same time model rather complex environments.

(2) Introduction of a flexible omega parameter.

(3) Direct application to a rather advanced VR task.

(4) The paper is extremely well written. It was a joy to read.

Weaknesses:

Almost none! In very few cases, the explanations could be a bit better.

Reviewer #3 (Public review):

Summary:

This paper aims to address the problem of exploring potentially rewarding environments that contain the danger, based on the assumption that an independent Pavlovian fear learning system can help guide an agent during exploratory behaviour such that it avoids severe danger. This is important given that otherwise later gains seem to outweigh early threats, and agents may end up putting themselves in danger when it is advisable not to do so.

The authors develop a computational model of exploratory behaviour that accounts for both instrumental and Pavlovian influences, combining the two according to uncertainty in the rewards. The result is that Pavlovian avoidance has a greater influence when the agent is uncertain about rewards.

Strengths:

The study does a thorough job of testing this model using both simulations and data from human participants performing an avoidance task. Simulations demonstrate that the model can produce "safe" behaviour, where the agent may not necessarily achieve the highest possible reward but ensures that losses are limited. Interestingly, the model appears to describe human avoidance behaviour in a task that tests for Pavlovian avoidance influences better than a model that doesn't adapt the balance between Pavlovian and instrumental based on uncertainty. The methods are robust, and generally, there is little to criticise about the study.

Weaknesses:

The extent of the testing in human participants is fairly limited but goes far enough to demonstrate that the model can account for human behaviour in an exemplar task. There are, however, some elements of the model that are unrealistic (for example, the fact that pre-training is required to select actions with a Pavlovian bias would require the agent to explore the environment initially and encounter a vast amount of danger in order to learn how to avoid the danger later). The description of the models is also a little difficult to parse.

Author response:

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

This paper provides a computational model of a synthetic task in which an agent needs to find a trajectory to a rewarding goal in a 2D-grid world, in which certain grid blocks incur a punishment. In a completely unrelated setup without explicit rewards, they then provide a model that explains data from an approach-avoidance experiment in which an agent needs to decide whether to approach or withdraw from, a jellyfish, in order to avoid a pain stimulus, with no explicit rewards. Both models include components that are labelled as Pavlovian; hence the authors argue that their data show that the brain uses a Pavlovian fear system in complex navigational and approach-avoid decisions. 

We thank the reviewer for their thoughtful comments. To clarify, the grid-world setup was used as a didactic tool/testbed to understand the interaction between Pavlovian and instrumental systems (lines 80-81) [Dayan et al., 2006], specifically in the context of safe exploration and learning. It helps us delineate the Pavlovian contributions during learning, which is key to understanding the safety-efficiency dilemma we highlight. This approach generates a hypothesis about outcome uncertainty-based arbitration between these systems, which we then test in the approach-withdrawal VR experiment based on foundational studies studying Pavlovian biases [Guitart-Masip et al., 2012, Cavanagh et al., 2013].

Although the VR task does not explicitly involve rewards, it provides a specific test of our hypothesis regarding flexible Pavlovian fear bias, similar to how others have tested flexible Pavlovian reward bias without involving punishments (e.g., Dorfman & Gershman, 2019). Both the simulation and VR experiment models are derived from the same theoretical framework and maintain algebraic mapping, differing only in task-specific adaptations (e.g., differing in action sets and temporal difference learning for multi-step decisions in the grid world vs. Rescorla-Wagner rule for single-step decisions in the VR task). This is also true for Dayan et al. [2006] who bridge Pavlovian bias in a Go-No Go task (negative auto-maintenance pecking task) and a grid world task. Therefore, we respectfully disagree that the two setups are completely unrelated and that both models include components merely labelled as Pavlovian.

We will rephrase parts of the manuscript to prevent the main message of our manuscript from being misconveyed. Particularly in the Methods and Discussion, to clarify that our main focus is on Pavlovian fear bias in safe exploration and learning (as also summarised by reviewers #2 and #3), rather than on its role in complex navigational decisions. We also acknowledge the need for future work to capture more sophisticated safe behaviours, such as escapes and sophisticated planning which span different aspects of the threat-imminence continuum [Mobbs et al., 2020], and we will highlight these as avenues for future research.

In the first setup, they simulate a model in which a component they label as Pavlovian learns about punishment in each grid block, whereas a Q-learner learns about the optimal path to the goal, using a scalar loss function for rewards and punishments. Pavlovian and Q-learning components are then weighed at each step to produce an action. Unsurprisingly, the authors find that including the Pavlovian component in the model reduces the cumulative punishment incurred, and this increases as the weight of the Pavlovian system increases. The paper does not explore to what extent increasing the punishment loss (while keeping reward loss constant) would lead to the same outcomes with a simpler model architecture, so any claim that the Pavlovian component is required for such a result is not justified by the modelling. 

Thank you for this comment. We acknowledge that our paper does not compare the Pavlovian fear system to a purely instrumental system with varying punishment sensitivity. Instead, our model assumes the coexistence of these two systems and demonstrates the emergent safety-efficiency trade-off from their interaction. It is possible that similar behaviours could be modelled using an instrumental system alone. In light of the reviewer’s comment, we will soften our claims regarding the necessity of the Pavlovian system, despite its known existence.

We also encourage the reviewer to consider the Pavlovian system as a biologically plausible implementation of punishment sensitivity. Unlike punishment sensitivity (scaling of the punishments), which has not been robustly mapped to neural substrates in fMRI studies, the neural substrates for the Pavlovian fear system (e.g., the limbic loop) are well known (see Supplementary Fig. 16).

Additionally, we point out that varying reward sensitivities while keeping punishment sensitivity constant allows our PAL agent to differentiate from an instrumental agent that combines reward and punishment into a single feedback signal. As highlighted in lines 136-140 and the T-maze experiment (Fig. 3 A, B, C), the Pavlovian system maintains fear responses even under high reward conditions, guiding withdrawal behaviour when necessary (e.g., ω = 0.9 or 1), which is not possible with a purely instrumental model if the punishment sensitivities are fixed. This is a fundamental point.

We will revise our discussion and results sections to reflect these clarifications.

In the second setup, an agent learns about punishments alone. "Pavlovian biases" have previously been demonstrated in this task (i.e. an overavoidance when the correct decision is to approach). The authors explore several models (all of which are dissimilar to the ones used in the first setup) to account for the Pavlovian biases. 

Thank you, we respectfully disagree with the statement that our models used in the experimental setup are dissimilar to the ones used in the first setup. Due to differences in the nature of the task setup, the action set differs, but the model equations and the theory are the same and align closely, as described in our response above. The only additional difference is the use of a baseline bias in human experiments and the RLDDM model, where we also model reaction times with drift rates which is not a behaviour often simulated in grid world simulations. We will improve our Methods section to ensure that model similarity is highlighted.

Strengths: 

Overall, the modelling exercises are interesting and relevant and incrementally expand the space of existing models. 

We thank reviewer #1 for acknowledging the relevance of our models in advancing the field. We would like to further highlight that, to the best of our knowledge, this is the first time reaction times in Pavlovian-Instrumental arbitration tasks have been modelled using RLDDM, which adds a novel dimension to our approach.

Weaknesses: 

I find the conclusions misleading, as they are not supported by the data. 

First, the similarity between the models used in the two setups appears to be more semantic than computational or biological. So it is unclear to me how the results can be integrated. 

We acknowledge the dissimilarity between the task setups (grid-world vs. approach-withdrawal). However, we believe these setups are computationally similar and may be biologically related, as suggested by prior work like Dayan et al. [2006], which integrates Go-No Go and grid-world tasks. Just as that work bridged findings in the appetitive domain, we aim to integrate our findings in the aversive domain. We will provide a more integrated interpretation in the discussion section of the revised manuscript.

Dayan, P., Niv, Y., Seymour, B., and Daw, N. D. (2006). The misbehavior of value and the discipline of the will. Neural networks, 19(8):1153–1160.

Secondly, the authors do not show "a computational advantage to maintaining a specific fear memory during exploratory decision-making" (as they claim in the abstract). Making such a claim would require showing an advantage in the first place. For the first setup, the simulation results will likely be replicated by a simple Q-learning model when scaling up the loss incurred for punishments, in which case the more complex model architecture would not confer an advantage. The second setup, in contrast, is so excessively artificial that even if a particular model conferred an advantage here, this is highly unlikely to translate into any real-world advantage for a biological agent. The experimental setup was developed to demonstrate the existence of Pavlovian biases, but it is not designed to conclusively investigate how they come about. In a nutshell, who in their right mind would touch a stinging jellyfish 88 times in a short period of time, as the subjects do on average in this task? Furthermore, in which real-life environment does withdrawal from a jellyfish lead to a sting, as in this task? 

Thank you for your feedback. As mentioned above, we invite the reviewer to potentially think of Pavlovian fear systems as a way how the brain might implement punishment sensitivity. Secondly, it provides a separate punishment memory that cannot be overwritten with higher rewards (see also Elfwing and Seymour 2017, and Wang et al, 2021)

Elfwing, S., & Seymour, B. (2017, September). Parallel reward and punishment control in humans and robots: Safe reinforcement learning using the MaxPain algorithm. In 2017 Joint IEEE International Conference on Development and Learning and Epigenetic Robotics (ICDL-EpiRob) (pp. 140-147). IEEE. 

Wang, J., Elfwing, S., & Uchibe, E. (2021). Modular deep reinforcement learning from reward and punishment for robot navigation. Neural Networks, 135, 115-126.

The simulation setups such as the following grid-worlds are common test-beds for algorithms in reinforcement learning [Sutton and Barto, 2018].

Any experimental setup faces the problem of having a constrained experiment designed to test and model a specific effect versus designing a lesser constrained exploratory experiment which is more difficult to model. Here we chose the former, building upon previous foundational experiments on Pavlovian bias in humans [Guitart-Masip et al., 2012, Cavanagh et al., 2013].  The condition where withdrawal from a jellyfish leads to a sting, though less realistic, was included for balancing the four cue-outcome conditions. Overall the task was designed to isolate the effect we wanted to test - Pavlovian fear bias in choices and reaction times, to the best of our ability. In a free operant task, it is very well likely that other components not included in our model could compete for control.

Crucially, simplistic models such as the present ones can easily solve specifically designed lab tasks with low dimensionality but they will fail in higher-dimensional settings. Biological behaviour in the face of threat is utterly complex and goes far beyond simplistic fight-flight-freeze distinctions (Evans et al., 2019). It would take a leap of faith to assume that human decision-making can be broken down into oversimplified sub-tasks of this sort (and if that were the case, this would require a meta-controller arbitrating the systems for all the sub-tasks, and this meta-controller would then struggle with the dimensionality j). 

We agree that safe behaviours, such as escapes, involve more sophisticated computations. We do not propose Pavlovian fear bias as the sole computation for safe behavior, but rather as one of many possible contributors. Knowing about the existence about the Pavlovian withdrawal bias, we simply study its possible contribution. We will include in our discussion that such behaviours likely occupy different parts of the threat-imminence continuum [Mobbs et al., 2020].

Dean Mobbs, Drew B Headley, Weilun Ding, and Peter Dayan. Space, time, and fear: survival computations along defensive circuits. Trends in cognitive sciences, 24(3):228–241, 2020.

On the face of it, the VR task provides higher "ecological validity" than previous screen-based tasks. However, in fact, it is only the visual stimulation that differs from a standard screen-based task, whereas the action space is exactly the same. As such, the benefit of VR does not become apparent, and its full potential is foregone. 

We thank the reviewer for their comment. We selected the action space to build on existing models [Guitart-Masip et al., 2012, Cavanagh et al., 2013] that capture Pavlovian biases and we also wanted to minimize participant movement for EEG data collection. Unfortunately, despite restricting movement to just the arm, the EEG data was still too noisy to lead to any substantial results. We will explore more free-operant paradigms in future works.

On the issue of the difference between VR and lab-based tasks, we note the reviewer's point. Note however that desktop monitor-based tasks lack the sensorimotor congruency between the action and the outcome. Second, it is also arguable, that the background context is important in fear conditioning, as it may help set the tone of the fear system to make aversive components easier to distinguish.

If the authors are convinced that their model can - then data from naturalistic approach-avoidance VR tasks is publicly available, e.g. (Sporrer et al., 2023), so this should be rather easy to prove or disprove. In summary, I am doubtful that the models have any relevance for real-life human decision-making. 

We thank the reviewers for their thoughtful inputs. We do not claim our model is the best fit for all naturalistic VR tasks, as they require multiple systems across the threat-imminence continuum [Mobbs et al., 2020] and are currently beyond the scope of the current work. However, we believe our findings on outcome-uncertainty-based arbitration of Pavlovian bias could inform future studies and may be relevant for testing differences in patients with mental disorders, as noted by reviewer #2. At a general level, it can be said that most well-controlled laboratory-based tasks need to bridge a sizeable gap to applicabilty in real-life naturalistic behaviour; although the principle of using carefully designed tasks to isolate individual factors is well established

Finally, the authors seem to make much broader claims that their models can solve safety-efficiency dilemmas. However, a combination of a Pavlovian bias and an instrumental learner (study 1) via a fixed linear weighting does not seem to be "safe" in any strict sense. This will lead to the agent making decisions leading to death when the promised reward is large enough (outside perhaps a very specific region of the parameter space). Would it not be more helpful to prune the decision tree according to a fixed threshold (Huys et al., 2012)? So, in a way, the model is useful for avoiding cumulatively excessive pain but not instantaneous destruction. As such, it is not clear what real-life situation is modelled here. 

We thank the reviewer for their comments and ideas. In our discussion lines 257-264, we discuss other works which identify similar safety-efficiency dilemmas, in different models. Here, we simply focus on the safety-efficiency trade-off arising from the interactions between Pavlovian and instrumental systems. It is important to note that the computational argument for the modular system with separate rewards and punishments explicitly protects (up to a point, of course) against large rewards leading to death because the Pavlovian fear response is not over-written by successful avoidance in recent experience. Note also that in animals, reward utility curves are typically convex. We will clarify this in the discussion section.

We completely agree that in certain scenarios, pruning decision trees could be more effective, especially with a model-based instrumental agent. Here we utilise a model-free instrumental agent, which leads to a simpler model - which is appreciated by some readers such as reviewer #2. Future work can incorporate model-based methods.

A final caveat regarding Study 1 is the use of a PH associability term as a surrogate for uncertainty. The authors argue that this term provides a good fit to fear-conditioned SCR but that is only true in comparison to simpler RW-type models. Literature using a broader model space suggests that a formal account of uncertainty could fit this conditioned response even better (Tzovara et al., 2018). 

We thank the reviewer for bringing this to our notice. We will discuss Tzovara et al., 2018 in our discussion in our revised manuscript.

Reviewer #2 (Public review): 

Summary: 

The authors tested the efficiency of a model combining Pavlovian fear valuation and instrumental valuation. This model is amenable to many behavioral decision and learning setups - some of which have been or will be designed to test differences in patients with mental disorders (e.g., anxiety disorder, OCD, etc.). 

Strengths: 

(1) Simplicity of the model which can at the same time model rather complex environments. 

(2) Introduction of a flexible omega parameter. 

(3) Direct application to a rather advanced VR task. 

(4) The paper is extremely well written. It was a joy to read. 

Weaknesses: 

Almost none! In very few cases, the explanations could be a bit better. 

We thank reviewer #2 for their positive feedback and thoughtful recommendations. We will ensure that, in our revision, we clarify the explanations in the few instances where they may not be sufficiently detailed, as noted.

Reviewer #3 (Public review): 

Summary: 

This paper aims to address the problem of exploring potentially rewarding environments that contain the danger, based on the assumption that an independent Pavlovian fear learning system can help guide an agent during exploratory behaviour such that it avoids severe danger. This is important given that otherwise later gains seem to outweigh early threats, and agents may end up putting themselves in danger when it is advisable not to do so. 

The authors develop a computational model of exploratory behaviour that accounts for both instrumental and Pavlovian influences, combining the two according to uncertainty in the rewards. The result is that Pavlovian avoidance has a greater influence when the agent is uncertain about rewards. 

Strengths: 

The study does a thorough job of testing this model using both simulations and data from human participants performing an avoidance task. Simulations demonstrate that the model can produce "safe" behaviour, where the agent may not necessarily achieve the highest possible reward but ensures that losses are limited. Interestingly, the model appears to describe human avoidance behaviour in a task that tests for Pavlovian avoidance influences better than a model that doesn't adapt the balance between Pavlovian and instrumental based on uncertainty. The methods are robust, and generally, there is little to criticise about the study. 

Weaknesses: 

The extent of the testing in human participants is fairly limited but goes far enough to demonstrate that the model can account for human behaviour in an exemplar task. There are, however, some elements of the model that are unrealistic (for example, the fact that pre-training is required to select actions with a Pavlovian bias would require the agent to explore the environment initially and encounter a vast amount of danger in order to learn how to avoid the danger later). The description of the models is also a little difficult to parse. 

We thank reviewer #3 for their thoughtful feedback and useful recommendations, which we will take into account while revising the manuscript.

We acknowledge the complexity of specifying Pavlovian bias in the grid world and appreciate the opportunity to elaborate on how this bias is modelled. In the human experiment, the withdrawal action is straightforwardly biased, as noted, while in the grid world, we assume a hardwired encoding of withdrawal actions for each state/grid. This innate encoding of withdrawal actions could be represented in the dPAG [Kim et. al., 2013]. We implement this bias using pre-training, which we assume would be a product of evolution. Alternatively, this could be interpreted as deriving from an appropriate value initialization where the gradient over initialized values determines the action bias. Such aversive value initialization, driving avoidance of novel and threatening stimuli, has been observed in the tail of the striatum in mice, which is hypothesized to function as a Pavlovian fear/threat learning system [Menegas et. al., 2018].

Additionally, we explored the possibility of learning the action bias on the fly by tracking additional punishment Q-values instead of pre-training, which produced similar cumulative pain and step plots. While this approach is redundant, and likely not how the brain operates, it demonstrates an alternative algorithm.

We thank the reviewer for pointing out these potentially unrealistic elements, and we will revise the manuscript to clarify and incorporate these explanations and improve the model descriptions.

Eun Joo Kim, Omer Horovitz, Blake A Pellman, Lancy Mimi Tan, Qiuling Li, Gal Richter-Levin, and Jeansok J Kim. Dorsal periaqueductal gray-amygdala pathway conveys both innate and learned fear responses in rats. Proceedings of the National Academy of Sciences, 110(36):14795–14800, 2013

William Menegas, Korleki Akiti, Ryunosuke Amo, Naoshige Uchida, and Mitsuko Watabe-Uchida. Dopamine neurons projecting to the posterior striatum reinforce avoidance of threatening stimuli. Nature neuroscience, 21(10): 1421–1430, 2018

eLife Assessment

The authors identify a novel relationship between exosome secretion and filopodia formation that has implications for cancer cell metastasis and neuronal synapse formation. Further, they identify the exosomal cargo, THSD7A, as a regulator of this process. The data presented is convincing, and represents an important advancement in our understanding of how these two biological processes are linked and play roles in regulating cell migration and cell-cell communication.

Reviewer #1 (Public review):

Summary:

The study significantly advances our understanding of how exosomes regulate filopodia formation. Filopodia play crucial roles in cell movement, polarization, directional sensing, and neuronal synapse formation. McAtee et al. demonstrated that exosomes, particularly those enriched with the protein THSD7A, play a pivotal role in promoting filopodia formation through Cdc42 in cancer cells and neurons. This discovery unveils a new extracellular mechanism through which cells can control their cytoskeletal dynamics and interaction with their surroundings. The study employs a combination of rescue experiments, live-cell imaging, cell culture, and proteomic analyses to thoroughly investigate the role of exosomes and THSD7A in filopodia formation in cancer cells and neurons. These findings offer valuable insights into fundamental biological processes of cell movement and communication and have potential implications for understanding cancer metastasis and neuronal development.

Weaknesses:

The conclusions of this study are in most cases supported by data, but some aspects of data analysis need to be better clarified and elaborated. Some conclusions need to be better stated and according to the data observed.

Reviewer #2 (Public review):

Summary:

The authors show that small EVs trigger the formation of filopodia in both cancer cells and neurons. They go on to show that two cargo proteins, endoglin, and THSD7A, are important for this process. This possibly occurs by activating the Rho-family GTPase CDC42.

Strengths:

The EV work is quite strong and convincing. The proteomics work is well executed and carefully analyzed. I was particularly impressed with the chick metastasis assay that added strong evidence of in vivo relevance.

Weaknesses:

The weakest part of the paper is the Cdc42 work at the end of the paper. It is incomplete and not terribly convincing. This part of the paper needs to be improved significantly

Reviewer #3 (Public review):

Summary:

The authors identify a novel relationship between exosome secretion and filopodia formation in cancer cells and neurons. They observe that multivesicular endosomes (MVE)-plasma membrane (PM) fusion is associated with filopodia formation in HT1080 cells and that MVEs are present in filopodia in primary neurons. Using overexpression and knockdown (KD) of Rab27/HRS in HT1080 cells, melanoma cells, and/or primary rat neurons, they found that decreasing exosome secretion reduces filopodia formation, while Rab27 overexpression leads to the opposite result. Furthermore, the decreased filopodia formation is rescued in the Rab27a/HRS KD melanoma cells by the addition of small extracellular vesicles (EVs) but not large EVs purified from control cells. The authors identify endoglin as a protein unique to small EVs secreted by cancer cells when compared to large EVs. KD of endoglin reduces filopodia formation and this is rescued by the addition of small EVs from control cells and not by small EVs from endoglin KD cells. Based on the role of filopodia in cancer metastasis, the authors then investigate the role of endoglin in cancer cell metastasis using a chick embryo model. They find that injection of endoglin KD HT1080 cells into chick embryos gives rise to less metastasis compared to control cells - a phenotype that is rescued by the co-injection of small EVs from control cells. Using quantitative mass spectrometry analysis, they find that thrombospondin type 1 domain containing 7a protein (THSD7A) is downregulated in small EVs from endoglin KD melanoma cells compared to those from control cells. They also report that THSD7A is more abundant in endoglin KD cell lysate compared to control HT1080 cells and less abundant in small EVs from endoglin KD cells compared to control cells, indicating a trafficking defect. Indeed, using immunofluorescence microscopy, the authors observe THSD7A-mScarlet accumulation in CD63-positive structures in endoglin KD HT1080 cells, compared to control cells. Finally, the authors determine that exosome-secreted THSD7A induces filopodia formation in a Cdc42-dependent mechanism.

Strengths:

(1) While exosomes are known to play a role in cell migration and autocrine signaling, the relationship between exosome secretion and the formation of filopodia is novel.

(2) The authors identify an exosomal cargo protein, THSD7A, which is essential for regulating this function.

(3) The data presented provide strong evidence of a role for endoglin in the trafficking of THSD7A in exosomes.

(4) The authors associate this process with functional significance in cancer cell metastasis and neurological synapse formation, both of which involve the formation of filopodia.

(5) The data are presented clearly, and their interpretation appropriately explains the context and significance of the findings.

Weaknesses:

(1) A better characterization of the nature of the small EV population is missing:

It is unclear why the authors chose to proceed to quantitative mass spectrometry with the bands in the Coomassie from size-separated EV samples, as there are other bands present in the small EV lane but not the large EV lane. This is important to clarify because it underlies how they were able to identify THSD7A as a unique regulator of exosome-mediated filopodia formation. Is there a reason why the total sample fractions were not compared? This would provide valuable information on the nature of the small and large EV populations.

(2) Data analysis and quantification should be performed with increased rigor:

a) Figure 1C - The optical and temporal resolution are insufficient to conclusively characterize the association between exosome secretion and filopodia. Specifically, the 10-second interval used in the image acquisitions is too close to the reported 20-second median time between exosome secretion and filopodia formation. Two-5 sec intervals should be used to validate this. It would also be important to correlate the percentage of filopodia events that co-occur with exosome secretion. Is this a phenomenon that occurs with most or only a small number of filopodia? Additionally, resolution with typical confocal microscopy is subpar for these analyses. TIRF microscopy would offer increased resolution to parse out secretion events. As the TIRF objective is listed in the Methods section, figure legends should mention which images were acquired using TIRF microscopy.

b) Figure 2 - It would be important to perform further analysis to concretely determine the relationship between exosome secretion and filopodia stability. Are secretion events correlated with the stability of filopodia? Is there a positive feedback loop that causes further filopodia stability and length with increased secretion? Furthermore, is there an association between the proximity of secretion with stability? Quantification of filopodia more objectively (# of filopodia/cell) would be helpful.

c) Figure 6 - Why use different gel conditions to detect THSD7A in small EVs from B16F1 cells vs HT1080 and neurons? Why are there two bands for THSD7A in panels C and E? It is difficult to appreciate the KD efficiency in E. The absence of a signal for THSD7A in the HT1080 shEng small EVs that show a signal for endoglin is surprising. The authors should provide rigorous quantification of the westerns from several independent experimental repeats.

(3) The study lacks data on the cellular distribution of endoglin and THSD7A:

a) Figure 6 - Is THSD7A expected to be present in the nucleus as shown in panel D (label D is missing in the Figure). It is not clear if this is observed in neurons. a Western of endogenous THSD7A on cell fractions would clarify this. The authors should further characterize the cellular distribution of THSD7A in both cell types. Similarly, the cellular distribution of endoglin in the cancer cells should be provided. This would help validate the proposed model in Figure 8.

b) Figure 7 - Although the western blot provides convincing evidence for the role of endoglin in THSD7A trafficking, the microscopy data lack resolution as well as key analyses. While differences between shSCR and shEng cells are clear visually, the insets appear to be zoomed digitally which decreases resolution and interferes with interpretation. It would be crucial to show the colocalization of endoglin and THSD7A within CD63-postive MVE structures. What are the structures in Figure 7E shSCR zoom1? It would be important to rule out that these are migrasomes using TSPAN4 staining. More information on how the analysis was conducted is needed (i.e. how extracellular areas were chosen and whether the images are representative of the larger population). A widefield image of shSCR and shEng cells and DAPI or HOECHST staining in the higher magnification images should be provided. Additionally, the authors should quantify the colocalization of external CD63 and mScarlet signals from many independently acquired images (as they did for the internal signals in panel F). Is there no external THSD7A signal in the shEng cells?

Author response:

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

The study significantly advances our understanding of how exosomes regulate filopodia formation. Filopodia play crucial roles in cell movement, polarization, directional sensing, and neuronal synapse formation. McAtee et al. demonstrated that exosomes, particularly those enriched with the protein THSD7A, play a pivotal role in promoting filopodia formation through Cdc42 in cancer cells and neurons. This discovery unveils a new extracellular mechanism through which cells can control their cytoskeletal dynamics and interaction with their surroundings. The study employs a combination of rescue experiments, live-cell imaging, cell culture, and proteomic analyses to thoroughly investigate the role of exosomes and THSD7A in filopodia formation in cancer cells and neurons. These findings offer valuable insights into fundamental biological processes of cell movement and communication and have potential implications for understanding cancer metastasis and neuronal development. 

Weaknesses: 

The conclusions of this study are in most cases supported by data, but some aspects of data analysis need to be better clarified and elaborated. Some conclusions need to be better stated and according to the data observed. 

We appreciate the reviewer's recognition of the impact of our study.  We will address the concerns about data analysis and statement of our conclusions in our full response to reviewers.

Reviewer #2 (Public review): 

Summary: 

The authors show that small EVs trigger the formation of filopodia in both cancer cells and neurons. They go on to show that two cargo proteins, endoglin, and THSD7A, are important for this process. This possibly occurs by activating the Rho-family GTPase CDC42. 

Strengths: 

The EV work is quite strong and convincing. The proteomics work is well executed and carefully analyzed. I was particularly impressed with the chick metastasis assay that added strong evidence of in vivo relevance. 

Weaknesses: 

The weakest part of the paper is the Cdc42 work at the end of the paper. It is incomplete and not terribly convincing. This part of the paper needs to be improved significantly.

We appreciate the reviewer's recognition of the impact of our study.  Indeed, more work needs to be done to clarify the role of Cdc42 in the induction of filopodia by exosome-associated THSD7A.  We anticipate that this will be a separate manuscript, delving in-depth into how exosome-associated THSD7A interacts with recipient cells to activate Cdc42 and carrying out a variety of assays for Cdc42 activation.

Reviewer #3 (Public review): 

Summary: 

The authors identify a novel relationship between exosome secretion and filopodia formation in cancer cells and neurons. They observe that multivesicular endosomes (MVE)-plasma membrane (PM) fusion is associated with filopodia formation in HT1080 cells and that MVEs are present in filopodia in primary neurons. Using overexpression and knockdown (KD) of Rab27/HRS in HT1080 cells, melanoma cells, and/or primary rat neurons, they found that decreasing exosome secretion reduces filopodia formation, while Rab27 overexpression leads to the opposite result. Furthermore, the decreased filopodia formation is rescued in the Rab27a/HRS KD melanoma cells by the addition of small extracellular vesicles (EVs) but not large EVs purified from control cells. The authors identify endoglin as a protein unique to small EVs secreted by cancer cells when compared to large EVs. KD of endoglin reduces filopodia formation and this is rescued by the addition of small EVs from control cells and not by small EVs from endoglin KD cells. Based on the role of filopodia in cancer metastasis, the authors then investigate the role of endoglin in cancer cell metastasis using a chick embryo model. They find that injection of endoglin KD HT1080 cells into chick embryos gives rise to less metastasis compared to control cells - a phenotype that is rescued by the co-injection of small EVs from control cells. Using quantitative mass spectrometry analysis, they find that thrombospondin type 1 domain containing 7a protein (THSD7A) is downregulated in small EVs from endoglin KD melanoma cells compared to those from control cells. They also report that THSD7A is more abundant in endoglin KD cell lysate compared to control HT1080 cells and less abundant in small EVs from endoglin KD cells compared to control cells, indicating a trafficking defect. Indeed, using immunofluorescence microscopy, the authors observe THSD7A-mScarlet accumulation in CD63-positive structures in endoglin KD HT1080 cells, compared to control cells. Finally, the authors determine that exosome-secreted THSD7A induces filopodia formation in a Cdc42-dependent mechanism. 

Strengths: 

(1) While exosomes are known to play a role in cell migration and autocrine signaling, the relationship between exosome secretion and the formation of filopodia is novel. 

(2) The authors identify an exosomal cargo protein, THSD7A, which is essential for regulating this function. 

(3) The data presented provide strong evidence of a role for endoglin in the trafficking of THSD7A in exosomes. 

(4) The authors associate this process with functional significance in cancer cell metastasis and neurological synapse formation, both of which involve the formation of filopodia. 

(5) The data are presented clearly, and their interpretation appropriately explains the context and significance of the findings. 

Weaknesses: 

(1) A better characterization of the nature of the small EV population is missing: 

It is unclear why the authors chose to proceed to quantitative mass spectrometry with the bands in the Coomassie from size-separated EV samples, as there are other bands present in the small EV lane but not the large EV lane. This is important to clarify because it underlies how they were able to identify THSD7A as a unique regulator of exosome-mediated filopodia formation. Is there a reason why the total sample fractions were not compared? This would provide valuable information on the nature of the small and large EV populations. 

We would like to clarify that there are two sets of proteomics data in the manuscript. The first was comparing bands from a Coomassie gel from two samples: small EVs and large EVs from B16F1 cells. In this proteomics experiment, we identified endoglin as present in small EVs, but not large EVs. For this experiment, we only sent 4 bands from the small EV lane, chosen based on their obvious banding pattern difference on the Coomassie gel.

In the second proteomics experiment, we used quantitative iTRAQ proteomics to compare small EVs purified from B16F1 control (shScr) and endoglin KD (shEng1 and shEng2) cell lines. In this experiment, we sent total protein extracted from small EV samples for analysis. So, these samples included the entire EV content, not just selected bands from a gel. In this experiment, we identified THSD7A as reduced in the shEng small EVs.

(2) Data analysis and quantification should be performed with increased rigor: 

a) Figure 1C - The optical and temporal resolution are insufficient to conclusively characterize the association between exosome secretion and filopodia. Specifically, the 10-second interval used in the image acquisitions is too close to the reported 20-second median time between exosome secretion and filopodia formation. Two-5 sec intervals should be used to validate this. It would also be important to correlate the percentage of filopodia events that co-occur with exosome secretion. Is this a phenomenon that occurs with most or only a small number of filopodia? Additionally, resolution with typical confocal microscopy is subpar for these analyses. TIRF microscopy would offer increased resolution to parse out secretion events. As the TIRF objective is listed in the Methods section, figure legends should mention which images were acquired using TIRF microscopy. 

We acknowledge that the frame rate naturally limits our estimates of the timing of filopodia formation after exosome secretion. We set out to show a relationship between exosome secretion and filopodia formation, based on their proximity in timing. While our data set shows a median time interval of 20 seconds, the true median could be between 10-30 seconds, based on our frame rate.  Regardless of the exact timing, our data show that exosome secretion is rapidly followed by filopodia formation events.

To address the question of the percentage of filopodia events that are preceded by exosome secretion, the reviewer is correct in stating that we might need TIRF microscopy to get an accurate calculation of this number.  Nonetheless, we will review our live imaging data for this experiment to determine if this calculation is possible. Again, we will be limited by the frame rate we used to capture the images, so we could possibly be missing secretion events taking place between the 10 second time intervals.  Regardless, for the secretion events that we visualized, we always observed subsequent filopodia formation.

No TIRF imaging was used in this manuscript.  A TIRF objective was used for selected neuron imaging (see methods); however, it was used for spinning disk confocal microscopy, not for TIRF imaging.  We will clarify this in the methods.

b) Figure 2 - It would be important to perform further analysis to concretely determine the relationship between exosome secretion and filopodia stability. Are secretion events correlated with the stability of filopodia? Is there a positive feedback loop that causes further filopodia stability and length with increased secretion? Furthermore, is there an association between the proximity of secretion with stability? Quantification of filopodia more objectively (# of filopodia/cell) would be helpful. 

Our data shows that manipulation of general exosome secretion, via Hrs knockdown, affects both de novo filopodia formation and filopodia stability (Fig 2g,h). Interestingly, knockdown of endoglin only affects de novo filopodia formation, while filopodia stability is unaffected (Fig 4g,h). These results suggest that filopodia stability is dependent upon exosome cargoes besides endoglin/THSD7A.  Such cargoes might include other extracellular matrix molecules, such as fibronectin. We previously showed that exosomes promote nascent cell adhesion and rapid cell migration, through exosome-bound fibronectin (Sung et al., Nature Communications, 6:7164, 2015). We also previously found that inhibition of exosome secretion affects the persistence of invadopodia, which are filopodia-dependent structures (Hoshino et al., Cell Reports, 5:1159-1168, 2013).  We agree that this is an interesting research direction, and perhaps future work could focus on exosomal factors that are responsible for filopodia persistence.

With regard to the way we plotted the filopodia data, we plotted the cancer cell data as filopodia per cell area so that it matched the neuron data, which was plotted as filopodia per 100 mm of dendrite distance. Since the neurons cannot be imaged as a whole cell, the quantification is based on the length of the dendrite in the image. We found that graphing the cancer cell data as filopodia per cell gave similar results as filopodia per cell area, as there were no significant differences in cell area between conditions and experiments. We plan to include a new supplementary figure showing the data in Figure 2 plotted as filopodia per cell to show that this quantification gives the same results.

c) Figure 6 - Why use different gel conditions to detect THSD7A in small EVs from B16F1 cells vs HT1080 and neurons? Why are there two bands for THSD7A in panels C and E? It is difficult to appreciate the KD efficiency in E. The absence of a signal for THSD7A in the HT1080 shEng small EVs that show a signal for endoglin is surprising. The authors should provide rigorous quantification of the westerns from several independent experimental repeats. 

Detection of THSD7A via Western blot was, unfortunately, not straightforward and simple. Due to the large size (~260 kDa) of THSD7A, its low level of expression in cancer cells, as well as the inconsistency of commercially available THSD7A antibodies, we had to troubleshoot multiple conditions.  We found that it was much easier to detect THSD7A in the human fibrosarcoma cell line HT1080 than in the mouse B16F1 cells, both in the cell lysates and in the small EVs. We were usually unable to detect THSD7A using these same conditions for the mouse melanoma B16F1 samples, but were successful using native gel conditions. We also detected THSD7A in rat primary neuron samples. All these samples were from different source organisms (human, mouse, rat) and from either cell lysates or extracellular vesicles, further complicating the analyses. Expression and maturation of THSD7A in these different cell types and compartments could involve different post-translational modifications, such as glycosylation, thus requiring different methods needed to detect THSD7A on Western blots and leading to different banding patterns. Based on our THSD7A trafficking data, we believe that in control cells, most of the THSD7A is getting trafficked and secreted via small EVs. As you can see in Figure 7A, the band for THSD7A in the shScr cell lysate is relatively light and also shows a double band similar to Figure 6E (both HT1080 samples).

With regard to the level of knockdown of THSD7A in the Western blot shown in Figure 6E, the normalized level is quantitated below the bands.  If you compare that quantitation to the filopodia phenotypes in the same panel, they are quite concordant.  Figures 7B and 7C show quantification of triplicate Western blots, highlighting the significant accumulation of THSD7A in shEng cell lysates, as well as significant small EV secretion of THSD7A in control and WT rescued conditions.

(3) The study lacks data on the cellular distribution of endoglin and THSD7A: 

a) Figure 6 - Is THSD7A expected to be present in the nucleus as shown in panel D (label D is missing in the Figure). It is not clear if this is observed in neurons. a Western of endogenous THSD7A on cell fractions would clarify this. The authors should further characterize the cellular distribution of THSD7A in both cell types. Similarly, the cellular distribution of endoglin in the cancer cells should be provided. This would help validate the proposed model in Figure 8. 

The image in figure 6D shows an HT1080 cell stained with phalloidin-Alexa Fluor 488 to visualize F-actin with or without expression of THSD7A-mScarlet.  In order to fully visualize the thin filopodia protrusions, the cellular plane of focus of the images for this panel was purposely taken at the bottom of the cell, where the cell is attached to the coverslip glass. Thus, we interpret the red signal across the cell body as THSD7A-mScarlet expression on the plasma membrane underneath the cell, not in the nucleus. The neuron images only include the dendrite portion of the neurons; therefore, there is no nucleus present in the neuronal images.

b) Figure 7 - Although the western blot provides convincing evidence for the role of endoglin in THSD7A trafficking, the microscopy data lack resolution as well as key analyses. While differences between shSCR and shEng cells are clear visually, the insets appear to be zoomed digitally which decreases resolution and interferes with interpretation. It would be crucial to show the colocalization of endoglin and THSD7A within CD63-postive MVE structures. What are the structures in Figure 7E shSCR zoom1? It would be important to rule out that these are migrasomes using TSPAN4 staining. More information on how the analysis was conducted is needed (i.e. how extracellular areas were chosen and whether the images are representative of the larger population). A widefield image of shSCR and shEng cells and DAPI or HOECHST staining in the higher magnification images should be provided. Additionally, the authors should quantify the colocalization of external CD63 and mScarlet signals from many independently acquired images (as they did for the internal signals in panel F). Is there no external THSD7A signal in the shEng cells? 

The images for Figure 7E were taken with high resolution on a confocal microscope.  Insets for Figure 7E were zoomed in so that readers could see the tiny structures.  Zoom 1 in Figure 7E shows areas of extracellular deposition. In these areas, we can see small punctate depositions that are positive for CD63 and/or THSD7A-mScarlet. Our interpretation of this staining is that the cells are secreting heterogeneous small EVs that are then attached to the glass coverslip. The images and zooms in Fig 7E were chosen to be representative and indeed reveal that there is more extracellular deposition of THSD7A-mScarlet outside the control shScr cells compared to the shEng cells, consistent with more export of THSD7A into small EVs from shScr cells when compared to those of shEng cells (Fig 7A,B). However, we did not quantify this difference, as these experiments were conducted with transient transfection of THSD7A-mScarlet and it is challenging to determine which cell the extracellular THSD7A-mScarlet came from, complicating any quantitative analysis on a per-cell basis.  Quantification of internal THSD7A localization is much more straightforward in this experimental regime.  Indeed, in Figure 7F we assessed internal colocalization of THSD7A-mScarlet and CD63, which we obtained by choosing only cells that were visually positive for THSD7A-mScarlet in each transient transfection and omitting all extracellular signals. Quantifying the extracellular colocalization of THSD7A and CD63 could certainly be a future direction for this project and would require establishing cells that stably express THSD7A-mScarlet.

Reporter John Dickerson talking about his notebook.

While he doesn't mention it, he's capturing the spirit of the commonplace book and the zettelkasten.

[...] I see my job as basically helping people see and to grab ahold of what's going on.

You can decide to do that the minute you sit down to start writing or you can just do it all the time. And by the time you get to writing you have a notebook full of stuff that can be used.

And it's not just about the thing you're writing about at that moment or the question you're going to ask that has to do with that week's event on Face the Nation on Sunday.

If you've been collecting all week long and wondering why a thing happens or making an observation about something and using that as a piece of color to explain the political process to somebody, then you've been doing your work before you ever sat down to do your work.

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Field Notes: Reporter's Notebook from Coudal Partners on Vimeo.

eLife Assessment

This valuable study investigates how the size of an LLM may influence its ability to model the human neural response to language recorded by ECoG. Overall, solid evidence is provided that larger language models can better predict the human ECoG response. Further discussion would be beneficial as to how the results can inform us about the brain or LLMs, especially about the new message that can be learned from this ECoG study beyond previous fMRI studies on the same topic. This study will be of interest to both neuroscientists and psychologists who work on language comprehension and computer scientists working on LLMs.

Reviewer #1 (Public review):

Summary:

The authors perform an analysis of the relationship between the size of an LMM and the predictive performance of an ECoG encoding model made using the representations from that LMM. They find a logarithmic relationship between model size and prediction performance, consistent with previous findings in fMRI. They additionally observe that as the model size increases, the location of the "peak" encoding performance typically moves further back into the model in terms of percent layer depth, an interesting result worthy of further analysis into these representations.

Strengths:

The evidence is quite convincing, consistent across model families, and complementary to other work in this field. This sort of analysis for ECoG is needed and supports the decade-long enduring trend of the "virtuous cycle" between neuroscience and AI research, where more powerful AI models have consistently yielded more effective predictions of responses in the brain. The lag analysis showing that optimal lags do not change with model size is a nice result using the higher temporal resolution of ECoG compared to other methods like fMRI.

Weaknesses:

I would have liked to have seen the data scaling trends explored a bit too, as this is somewhat analogous to the main scaling results. While better performance with more data might be unsurprising, showing good data scaling would be a strong and useful justification for additional data collection in the field, especially given the extremely limited amount of existing language ECoG data. I realize that the data here is somewhat limited (only 30 minutes per subject), but authors could still in principle train models on subsets of this data.

Separately, it would be nice to have better justification of some of these trends, in particular the peak layerwise encoding performance trend and the overall upside-down U-trend of encoding performance across layers more generally. There is clearly something very fundamental going on here, about the nature of abstraction patterns in LLMs and in the brain, and this result points to that. I don't see the lack of justification here as a critical issue, but the paper would certainly be better with some theoretical explanation for why this might be the case.

Lastly, I would have wanted to see a similar analysis here done for audio encoding models using Whisper or WavLM as this is the modality where you might see real differences between ECoG and other slower scanning approaches. Again, I do not see this omission as a fundamental issue, but it does seem like the sort of analysis for which the higher temporal resolution of ECoG might grant some deeper insight.

Reviewer #2 (Public review):

Summary:

This paper investigates whether large language models (LLMs) of increasing size more accurately align with brain activity during naturalistic language comprehension. The authors extracted word embeddings from LLMs for each word in a 30-minute story and regressed them against electrocorticography (ECoG) activity time-locked to each word as participants listened to the story. The findings reveal that larger LLMs more effectively predict ECoG activity, reflecting the scaling laws observed in other natural language processing tasks.

Strengths:

(1) The study compared model activity with ECoG recordings, which offer much better temporal resolution than other neuroimaging methods, allowing for the examination of model encoding performance across various lags relative to word onset.

(2) The range of LLMs tested is comprehensive, spanning from 82 million to 70 billion parameters. This serves as a valuable reference for researchers selecting LLMs for brain encoding and decoding studies.

(3) The regression methods used are well-established in prior research, and the results demonstrate a convincing scaling law for the brain encoding ability of LLMs. The consistency of these results after PCA dimensionality reduction further supports the claim.

Weaknesses:

(1) Some claims of the paper are less convincing. The authors suggested that "scaling could be a property that the human brain, similar to LLMs, can utilize to enhance performance", however, many other animals have brains with more neurons than the human brain, making it unlikely that simple scaling alone leads to better language performance. Additionally, the authors claim that their results show 'larger models better predict the structure of natural language.' However, it remains unclear to what extent the embeddings of LLMs capture the "structure" of language better than the lexical semantics of language.

(2) The study lacks control LLMs with randomly initialized weights and control regressors, such as word frequency and phonetic features of speech, making it unclear what the baseline is for the model-brain correlation.

(3) The finding that peak encoding performance tends to occur in relatively earlier layers in larger models is somewhat surprising and requires further explanation. Since more layers mean more parameters, if the later layers diverge from language processing in the brain, it raises the question of what aspects of the larger models make them more brain-like.

Reviewer #3 (Public review):

This manuscript studies the connection between neural activity collected through electrocorticography and hidden vector representations from autoregressive language models, with the specific aim of studying the influence of language model size on this connection. Neural activity was measured from subjects who listened to a segment from a podcast, and the representations from language models were calculated using the written transcription as the input text. The ability of vector representations to predict neural activity was evaluated using 10-fold cross-validation with ridge regression models.

The main results are that (as well summarized in section headings):

(1) Larger models predict neural activity better.

(2) The ability of language model representations to predict neural activity differs across electrodes and brain regions.

(3) The layer that best predicts neural activity differs according to model size, with the "SMALL" model showing a correspondence between layer number and the language processing hierarchy.

(4) There seems to be a similar relationship between the time lag and the ability of language model representations to predict neural activity across models.

Strengths:

(1) The experimental and modeling protocols generally seem solid, which yielded results that answer the authors' primary research question.

(2) Electrocorticography data is especially hard to collect, so these results make a nice addition to recent functional magnetic resonance imaging studies.

Weaknesses:

(1) The interpretation of some results seems unjustified, although this may just be a presentational issue.

a) Figure 2B: The authors interpret the results as "a plateau in the maximal encoding performance," when some readers might interpret this rather as a decline after 13 billion parameters. Can this be further supported by a significance test like that shown in Figure 4B?

b) Figure S1A: It looks like the drop in PCA max correlation is larger for larger models, which may suggest to some readers that the same trend observed for ridge max correlation may not hold, contra the authors' claim that all results replicate. Why not include a similar figure as Figure 2B as part of Figure S1?

(2) Discussion of what might be driving the main result about the influence of model size appears to be missing (cf. the authors aim to provide an explanation of what seems to drive the influence of the layer location in Paragraph 3 of the Discussion section). What explanations have been proposed in the previous functional magnetic resonance imaging studies? Do those explanations also hold in the context of this study?

(3) The GloVe-based selection of language-sensitive electrodes (at least to me) isn't explained/motivated clearly enough (I think a more detailed explanation should be included in the Materials and Methods section). If the electrodes are selected based on GloVe embeddings, then isn't the main experiment just showing that representations from larger language models track more closely with GloVe embeddings? What justifies this methodology?

(4) (Minor weakness) The main experiments are largely replications of previous functional magnetic resonance imaging studies, with the exception of the one lag-based analysis. Is there anything else that the electrocorticography data can reveal that functional magnetic resonance imaging data can't?

Author response:

We thank the reviewers for their thoughtful feedback and valuable comments. We plan to fully address their concerns by including the following experiments and analyses:

Reviewer 1 suggested exploring data scaling trends for encoding models, as successful scaling would justify larger datasets for language ECoG studies. To estimate scaling effects, we will develop encoding models on subsets of our data.

Reviewer 2 expressed uncertainty about the baseline for model-brain correlation and recommended adding control LLMs with randomly initialized weights. In response, we will generate embeddings using untrained LLMs to establish a more robust baseline for encoding results.

Reviewer 2 also proposed incorporating control regressors such as word frequency and phonetic features of speech. We will re-run our modeling analysis using control regressors for word frequency, 8 syntactic features (e.g., part of speech, dependency, prefix/suffix), and 3 phonetic features (e.g., phonemes, place/manner of articulation) to assess how much these features contribute to encoding performance.

Reviewer 3 raised concerns that the “plateau in maximal encoding performance” was actually a decline for the largest models. We will add significance tests in Figure 2B to clarify this issue.

Reviewer 3 also noted that in Supplementary Figure 1A, the decline in encoding performance was more pronounced when using PCA to reduce embedding dimensionality, in contrast to the trend observed when using ridge regression. To address this, we will attempt to replicate the observed scaling trends in Figure 2B using PCA combined with OLS.

Additionally, we will provide a point-by-point response and revise the manuscript with updated analyses and figures in the near future.

Paul Pham shares an interesting take on how expectations play out in schools. He points out that if a student from a different background, like a Latina/o, joins a mostly Asian class or activity, they often get a lot of positive attention just for being there. While Paul sees this as a good thing, there’s a deeper issue tied to Claude Steele’s idea of disidentification. In this view, if a non-Asian student joins an AP class or a tough extracurricular, they might be greeted with surprise and praise for doing something unexpected. This reinforces the stereotype that they’re exceeding low expectations. This stigma can make them feel disengaged from academics unless they have mentors or support that recognize and encourage their abilities. It really shows how crucial it is to challenge these stereotypes and create a more welcoming environment for everyone.

While many schools are starting to focus more on students' mental health and offering counseling services, the counselors often don’t really know how to help students of color. Violet, who is multiracial Asian American and part Latino, shares how she feels invisible in her mostly white community and often gets lumped together with Mexican Americans. It shows a real lack of understanding about her identity. When a white counselor tried to help by taking students on just one tour of a local community college, it felt pretty shallow and didn’t tackle the real issues they face. It really emphasizes how important it is for counselors to understand and be sensitive to the unique experiences of diverse students.

When Asian American students try to speak up about bullying, they often get told to “thicken their skin” and not take it seriously, while the kids making fun of them face little to no consequences. Parents worry about complaining because they’re afraid it’ll just cause more trouble or make things worse. Even though schools are pushing for multiculturalism and celebrating diversity, sometimes it feels pretty surface-level. This “happy” approach can blind teachers and administrators to the real effects of racism on students of color and the bigger problem of institutional racism in schools. It’s frustrating to see how these attitudes can really undermine the experiences of students who are struggling.

I think it highlights the mixed messages about how Asian Americans are seen. On one side, they’re often labeled as the “model minority,” seen as super successful and even “whiter than whites.” But at the same time, they deal with serious racism and discrimination, facing barriers in education and jobs, plus real threats of violence—similar to what Black communities experience. Gary Okihiro points out that this puts Asians in a tricky spot within the larger black-and-white racial landscape, where both groups face their own kinds of oppression. It really shows how complex these issues are and how important it is to understand the different ways racial struggles affect Asian Americans.

It really shows how harmful stereotypes about Asian Americans are still everywhere in media. Cartoons like "Mr. Wong" feature ridiculous, over-the-top characters that just feed into negative views, and some people think it’s just a joke, which is super frustrating. Movies like "Team America: World Police" do the same thing, piling on racial mockery and telling anyone who’s offended to lighten up. Even shows like "Desperate Housewives" join in with lines that reinforce stereotypes, like questioning a doctor’s background just because of where they’re from. It’s disappointing that this kind of stuff is still considered okay in mainstream media, and it’s awesome to see Asian and Pacific Islander communities standing up against it.

This paragraph emphasizes the stress and pain that come from dealing with racism and stereotypes. The "model minority" label, often used by those in power, creates a lot of pressure to fit into a white-dominated culture, which can be really tough. It highlights how these expectations can lead to mental health struggles, making it clear that it’s more about the external pressures than anything inherent in the community.

very informative Thanks atman for such Information

这不是很显然的吗？

Parth’s annotation on this line introduces an interesting interpretation of its origins in Antony and Cleopatra. He identifies that “this line is an imitation of a line we see early on in Antony and Cleopatra—with one change: the word "barge" is changed to a capitalized "Chair" here…Chair might not be referring to a literal chair, which isn't a proper noun—but an organizational position. A "Chair" in a company is an executive position; likewise, a metaphorical Chair in a kingdom may refer to one's supreme status” (Jain). I’d like to expand the scope of the Antony and Cleopatra reference beyond specific lines that Eliot incorporated into “The Waste Land,” now through Parth’s lens of Cleopatra’s status and power within the play, her “Chair” within Antony and Cleopatra, and therefore “The Waste Land” as well. When reading Antony and Cleopatra, I couldn’t help but take note of the many ways in which Cleopatra both adopts the role of the queen in a literal game of chess. In a very broad sense, Cleopatra’s decision to fake her own suicide, to temp Antony with her death, is quite analogous to the physical and strategic features of the queen on a chess board. For example, while Cleopatra plots to win the attention of Antony, she says, “I have nothing Of woman in me: now from head to foot // I am marble-constant” (Shakespeare, V.II). In portraying herself as hard as marble, she renders herself part of a chess set, as they are often made of marble. The image of a chess board is then transferred to the marble as it appears here in “The Waste Land,” further convincing me that Cleopatra is the queen of Eliot’s chess set as it is understood in “A Game of Chess.” Additionally, Cleopatra embodies the characteristics of a chess piece as a powerful strategic asset when used correctly. She even says, “come hither, come! come, come, and take a queen worth many babes and beggars” (Shakespeare, V.II). “Take” is the terminology in a game of chess for the capture of a piece. I believe it’s possible that both Shakespeare and Eliot recognized the parallels between Cleopatra’s actions and the act of sacrificing a piece in the game of chess. However, Cleopatra’s move was in vain, and ultimately, when she sacrifices her life, amounts to nothing. In other words, the power she possessed through her “Chair” is only valuable insofar as it is spent to win the Game of Chess. Meanwhile, the woman in “The Waste Land” also sits dead on her throne, her status reduced, presumably having met a similarly wasteful fate.

I would like to elaborate on Quisha’s point in her annotation for this section. She investigates the similarities between the original epigraph from Heart of Darkness, and the one from Ovid in the final draft, stating that the Sybil “deems her body as if it is a piece of waste. Kurtz is similar – he is ill; his health in a wasted state. The moment before his death he, too, is filled with superfluous knowledge; yet he cannot make use of it for death soon transcends him. The two epigraphs do resemble each other in many ways, and I'm curious about the exact reason as to why Eliot chose the one of Sybil over Kurtz” (Lee). I think she does something really interesting here by connecting the title of the poem to the epigraph, which is further supported by Weston’s way of connecting the physical condition of a person to the state of the land itself. This suggests that Eliot intended for Kurtz, and later the Sybil, to embody the Waste Land, the poem itself. That said, I believe the Sybil epigraph actually accomplishes this in a more complete way, which is why Eliot chose it.

To begin, Eliot’s original title for “The Waste Land” was “He Do the Police in Different Voices,” from Dickens’ Our Mutual Friend. We see here the origins of the polyphony of different voices throughout the poem–in the final draft, the multitude of voices essentially constitutes the poem itself. When Eliot swapped this title for something that didn’t directly relate to the “Different Voices,” He likely sought to compensate using the epigraph. Like the Sybil, Kurtz wishes to die. He says, “I am lying here in the dark waiting for death” (Conrad, 3). However, unlike the Sybil, when he is on the brink of death, he actually loses his voice: “he cried out twice, a cry that was no more than a whisper” (Conrad, 3). Considering the time Eliot spends alluding to various hellscapes and states of death throughout the poem, it simply does not suit the form of the poem if the establishing character were to lose their voice in death, or, as Quisa pointed out, when their body becomes a waste land.

The Sybil, on the other hand, serves Eliot’s purpose beautifully. She, too, wishes for death, perpetually suffering as she wastes away in this in-between state. However, significantly she retains her voice. As she leans closer and closer to true death, more and more of her physical being deteriorates. Not only is her voice one of her defining characteristics, but if her body is “The Waste Land,” is Eliot’s poem itself, then it too assumes an perpetually shrinking quality. Her physical form grows smaller and smaller, approaching a state of nothingness, but (for the purposes of this investigation) never reaching it. Mathematically, an infinitely diminishing quantity is considered to approach zero. Zero is a very difficult number to define in the realm of reality. It is nothing, yet it is nothing in a pluralistic sense–it is “not any,” but that is not the same as “not one” or “not 10.” If the Sybil and her voice, and therefore “The Waste Land” and its voice, can be defined as zero, we see that The Sybil successfully represents the indefinite cacophony of the voices of the poem, as well as its tendency towards a state of nothingness.

While the “Death by Water” section of “The Waste Land” is seemingly short, Eliot’s original draft was actually quite long. In this draft, Eliot evokes multiple concepts that I understand as foundational to understanding the published version. Ultimately, however, I want to explore a sense of penetrability and the impenetrability throughout “Death By Water”. One of the first points in which the notion of impenetrability appears is in Eliot’s original draft when the narrator speaks of the horizon. They describe the horizon as, “a long white line, a wall, a barrier”, which suggests a sense of the horizon as infinite and impenetrable, especially in the sense that it is unconquerable for sailors. Another aspect of impenetrability in sailing is the ship itself, which floats on water that constantly tries to get in. Additionally, the sails, inferred by the mentions of wind, are also impenetrable to the forces of nature, such as the wind that is actually harnessed to achieve human goals. Thus, we can understand this impenetrability as representative of humanities disconnect from the wider world itself, which constantly seeks to enter and conquer “humanity”. Another important mention of penetration from the draft can be seen with Eliot’s multiple references to the sailors drinking alcohol. Through drinking, the sailors try to forget their anguish at sea by thinking about “home and dollars and the pleasant violin//At Arm Brown’s joint, and the girls and the gin”. Thus, the sailors are allowing their bodies (which are generally impenetrable by liquid), to consume alcohol, a poison, in order to escape their pain. And so, we are met with a paradox as Sailors become more vulnerable and penetrable to liquid in order to escape pain. When we arrive at the published version of Eliot’s poem (which appears at the very end of his draft), Phlebas the sailor is described as drowned. On line 317, Eliot describes how “a current under sea // Picked his bones in whispers”. Again, we return to the impenetrable human body, which when drowning, becomes penetrated by water, and is reduced to nothing but bones, which are not only penetrable but completely submerged in water themselves. Regarding the previous mentioning of drinking, we can interpret drowning as an ultimate form of becoming penetrated as a method of escapism. Interestingly, this escapism is an allowing of the previously disconnect nature to penetrate the human self, or “humanity”.

These lines of “The Waste Land” can be traced back to Dante’s Inferno, specifically Purgatorio, or Purgatory. Translated to English, Dante’s text reads “please remember me, who am La Pia. Siena made me, in Maremma I was undone.” The mention of Italian cities such as Siena and Maremma and the state of being undone clearly connects with Eliot’s mentioning of Richmond and Kew, also cities, undoing the narrator. Thus, we can infer that Eliot intended to connect the “trams and dusty trees” to Purgatory, a realm essentially between hell and heaven. The use of trams, interestingly, expanded from carrying passengers to carrying war cargo and industrial workers during the Great War and industrialism. Alongside these roles of trams, the “dusty trees” seem to suggest ash or dust from factories or explosions, also tying into industrialism and war. Thus, we can understand the once innocent trams and trees as tainted by a world of destructive progress and violence, forcing them into a middle state similar to purgatory. Additionally, the notion of the narrator lying supine, or back against the floor, in a narrow canoe, introduces another tension between the narrator and the water around him. While he is unable to move the canoe, and the water essentially guides it, he is still in this impenetrable man-made vessel, able to be on the level with the water, but not in it. Thus, the reader can interpret a sense of anxiety, which is exacerbated by the notion of being “undone” by the cities of Richmond and Kew. Interestingly, the river Thames, which is previously mentioned in this poem, is the only river that connects Richmond and Kew. As for the “undoing” of the narrator by the cities, one can interpret the narrator’s frustration and imprisonment in the canoe as spiritually degrading, especially as they pass cities bustling with culture and people who are able to move about. The narrator’s inescapable isolation from these cities portrays a contrast between nature and humanity, in which the narrator finds themselves in a middle-ground as well.

Eliot’s interest in death by suicide (as we see in the Sybil epigraph) is brought to life here in an overlap of Shakesphere references. Of course, the first line here is an echo of the Tempest reference in Burial of the Dead. Notably, it is a reference to Alonso, who tried to drown himself upon hearing false news of his son’s death. Then, the second line is quite feasibly a reworking of the famous, “To be, or not to be,” given that they are syntactically analogous with the placement of commas. Actually, they are structurally inverted, with the Hamlet comma placed in between the second and third syllable (x x, x x x x), while Eliot’s comma is placed in between the fourth and fifth syllable (x x x x, x x). Hamlet’s line is most popularly interpreted as a contemplation of suicide. Together, these two references seem to point to Ophelia’s death, and suggest Eliot decides that hers was a death by suicide as well.

The way Eliot inverts the structure of Hamlet’s line brings to mind the concept of a reflection, such as the “glassy stream” in which Ophelia drowns, as if the Eliot line floats on the surface of a body of water. If we were to indulge that imagery, it certainly would reaffirm the ideas of death and corpses, seeing as corpses float in water. This calls to mind the line “fear death by water” from Burial of the Dead in the tarot section. Perhaps Eliot means to prophesize suicidal drowning, which adds an interesting wrinkle to the grave motif that we’ve seen so often up to this point: does Eliot’s focus shift from the ground as a resting place to that of a water-submerged grave in A Game of Chess?

I am interested in the indentation of “I sat upon the shore” in a section that is otherwise left-justified. The visual effect of the standalone line in the left-aligned stanza mimics a cliff / shore situation where a fisher can lower their hook. The “shore” is not supported by any words in the line directly beneath it, just as the sand underneath the waves sift under compression, shapeshifting and fluid. Extending this literal interpretation, what we find on the other end of the line is the subterranean substance beneath the shore’s surface. Our last line, “Shantih shantih shantih” is also indented, although not as much as the first line is. This is a call and response that not only sandwiches the mixture of content (similar yet unidentical as the shifting sands) with visibly-identifiable structure but also clarifies the mission the narrator has set out to achieve: finding peace.

Nautical imagery is not limited to semantics, however. Beyond fishing on shores and London Bridge collapsing (into the River Thames), the spacing of closing line “Shantih shantih shantih” vaguely resembles the ebb and flow of waves departing shore. I am puzzled by the alignment of these last three words, and one justification (haha because it’s not left-justified) I concluded is that each “shanih” corresponds to a moment in time, with the subject “I” in the first line denoting the present. The first, capitalized “shantih” is for the past - a violent amalgamation of tragedies spanning centuries, mythologies, and even languages, yet the narrator still possesses the burgeoning hope to pray for peace. The second is for the present - a conflicted narrative between “Fishing”, present participle, and “have shored”, present perfect tense, an active search for reconciliation. And the last is for the future - nebulous with a promise of revenge, for “Hieronymo’s mad againe”. What strikes me aside from Eliot’s refusal to spell alluded character names correctly is the simultaneous looming and absence of destiny. A final prayer for peace suggests the future may need all the divine intervention it can get. The residual aggression from the Spanish Tragedy, which in TWL, is the universal tragedy, lingers in the falling infrastructure and human decay. Yet, the future is markedly absent throughout the stanza. The subject is positioned “with the arid plain behind me”. This direction acknowledges the past and deems it infertile. But what lies ahead? What of the future? It is unwritten, unpunctualized, and utterly neglected.

Combining the Tarot-reading interpretation of the poem’s end and my earlier theory of fishing (if you were to draw a line between the “e” of “shore” and the “h” of the last“shantih”, you see a fishing line attached to a hook. If you really squint), we realize Eliot propels the reader into The Waste Land, or rather, he brings the waste land to us. We are all on our own holy grail quests, fishing for peace.

One aspect of Eliot’s description of the mountains in “What the Thunder Said” is the idea of the inability to exist or be present in a space. Eliot describes how “Here one can neither stand nor lie nor sit”, addressing what could be argued to be the main forms of human existence in rest. The idea of standing suggests a pause in walking, and thus not being able to stand suggests a continuous journey. The idea of sitting refers to a time of relaxation or restoration where one is still active in the body as well as the mind. This state of being is also impossible in the mountains. Finally, the notion of lying invites the possibility of sleep, collapse, and death. However, as the narrator claims, one cannot enter such a state in the mountains either. The paradoxical banning of sleep or death in the barren mountains evokes the previously mentioned story of Sybil, who is unable to die and escape her immortality. In this understanding of constant progression and movement, one finds themselves in a state of inescapable vitality. This idea of restlessness and vitality also appears in one of Eliot’s sources, a poem named “What the Thrush Said”, by John Keat. In this poem, Keat describes how “He who saddens//At thought of idleness cannot be idle//And he’s awake who thinks himself asleep.” Again, the reader is met with the notion not being able to be idle, but interesting, Keat’s poem also associates this state of restlessness with the human mind and consciousness. As one sleeps, they typically don’t realize they are sleeping, and as Keat describes, the realization that one is asleep is a state of awareness. I also believe we can interpret these descriptions of physical inabilities to rest as applicable the mental realm, specifically in the case of anguish. The inability to rest, stop, or even recover through sleep seems representative of mental torment, which, of course, has been discussed through many stories throughout “The Waste Land”. This endless vitality and restlessness of the mind can also be seen as the underlying force behind the fractured and constantly shifting essence of the poem, especially in terms of Eliot’s formation of dialogue and descriptions.

I think there is a strong relationship between poverty and education. Poverty can impact child’s ability to succeed in school. The educators and policymakers should design strategies to help economically disadvantaged students to ensure all students, regardless of their socioeconomic background, receive support in various aspects. There is no excuse for policymaker not to provides sufficient needs for all students.

Poverty, in my opinion, is the struggle of those who lack the means to improve their lot in life. Even though many people struggle, it might be difficult to succeed and get by. Fortunately, those living in poverty have access to opportunities provided by schools and scholarships, enabling them to improve their lives and attend prestigious universities across the globe.

While the father is clearly moving in a positive direction in terms of acceptance of his son's uniqueness, the fact that it took this long for him to appreciate his son is concerning, and I struggle to believe that there aren't any more underlying issues. It's a very significant change going from occasionally wishing your son would disappear to being amazed by him, and while it's positive, the fact that those initial thoughts even existed in the first place is troubling.

This passage stuck out to me because I had to reread it a few times before realizing it was referring to a blind girl, and I thought the way in which Kaden perceived this girl and her cane as a wand was beautiful

I understand where he is coming from, but I still struggle to get behind the idea that he has wished that his child would disappear.

I find this so disturbing. I can't believe a father would ever feel this way about his own son.

I still struggle to understand why Martin was so unhappy about Sebastian being able to stand. I don't see why it would be such a problem.

The fact that the father wasn't immediately happy upon seeing his child standing shows that he may have an underlying ableist attitude toward his son.

I am not sure why, but this line seems dehumanizing to me

The fact that Martin didn't call the police and get as much help as possible because he wasn't sure how to describe his son struck me because I would expect a father to do whatever he could possible to find his missing child

This fear of the mother's may reflect the fears that parents of children with disabilities may often face in regards to others seeing them as different

The playpen is described as a "hurdle" suggesting that the baby likely felt the need to escape the parents' constant arguments

The image of a baby jumping over the rail of a playpen and running away surpasses typical infant abilities as most babies aren't even capable of walking until they are one, suggesting that the child’s unique body makes him extraordinary

When trolling is used to illustrate fraud, it can be beneficial and promote critical thinking. However, when used to harass or bully someone, it can also be harmful and cause mental pain. The purpose of trolling and how it affects the targeted people or communities determine the result.

Stereotypes can be a hard thing to ethically judge. One should always leave room for humility in their understandings of other people. So much wrong about this world is the stubbornness and unwillingness to learn about others and see where an individual is coming from. At the same time, generalizations (like the one I just made) can be useful tools that at the very least hold partial/likely truths. In this way, they are like risky shortcuts; they can be useful if you are willing to take a few steps backwards should you find yourself in the wrong.

Problem #2の変数可視化表

To handle trolling, it's best to ignore and avoid engaging, as trolls thrive on attention. Users can use platform tools to block or report trolls, keeping the online environment controlled and safe.

I think the real issue here is treating "don't feed the trolls" as a maxim. Of course it is wrong to fault victims, but there is a big difference between assigning fault and offering a strategy to be employed. It won't always work, but it is an action one can take to attempt to minimize their personal suffering while waiting for a more ideal change in the system.

Problem #4の変数可視化表

I think this is what AI should be. I think that it makes most sense for humans to live in a world where AI exists this way. What is wrong with AI remaining this way? If it pretends to reciprocate why is real consciousness needed?

I don't think that these robots should deserve rights. They are real humans or Americans that are protected by the U.S. Constitution. Again, I think if Americans had to treat Ai as if it were a real U.S. citizen it may bring more harm than good.

I think that abiding ethical morals, is more important than having AI that has sentience. With it's advancements I think it imposes more on real humans' lives and it would probably do more harm than good if it had genuine emotions and consciousness.

If the current AI realm is what used to see as science-fiction, and consciousness is within the near future, then is what is "out of reach" going to become a reality in the future?

The author is right people are already unsure about how to treat animals, fetuses, and others, so adding AI could make things even more confusing. We should figure out our current ethical problems before worrying about how to treat AI.

The Design Policy of the Excluded Middle could hold back AI research by preventing us from creating systems that might help us learn about real sentience. How can we be careful with ethics while still trying out new AI ideas?

Shouldn’t we focus on improving the treatment of known sentient beings before worrying about the moral status of potential sentient AI?

If a sentient AI like Robot Alpha existed, it would make us rethink how do we treat AI? It would be the same as a person or maybe you'd think of it as a pet or something.

How so? The author should have explained this answer in better detail.

I don't think AI will ever be a sentient or alive being

the freedom came with something she was not free of controlling

For an inexpensive starter machine ($5-25) that's easy to find, easy to get parts for and has a reasonable chance of working when in "unknown" or "untested" condition, I'd recommend one of the following ubiquitous, but solid machines which show up almost daily on ShopGoodwill.com:

• Royal Quiet De Luxe https://typewriterdatabase.com/Royal.Quiet+De+Luxe.72.bmys
• Remington Quiet Riter https://typewriterdatabase.com/Remington.Quiet-Riter.42.bmys
• Smith-Corona Clipper (their Sterling, Silent, and Silent Super are very similar, but can sometimes be more expensive; they often have a few more features like tabs, but are otherwise the same machine) https://typewriterdatabase.com/Smith+Corona.Clipper.86.bmys

They'd all make excellent starter machines for a younger kid. The black models with glass keys from the 1940s will look a bit more old school/classic while the more industrial browns and grays with plastic keys from the 1950s are still solid choices. You might also find some later 60s/70s versions of these machines (or variations), and while they may be a bit more colorful, they'll usually have a lot more cheap plastic and can potentially have cheaper builds. (My parents got me my first typewriter, a 1948 Smith-Corona Clipper, in the mid-1980s when I was 10—I have it today and it still works as well as it did then; I still also love the airplane on the hood.)

If you want something simple with a bit of color you can also look at the 70s/80s Brother Charger 11 which is pretty ubiquitious and inexpensive as well.

Since you have some time, you can wait for one in better looking cosmetic condition (and with a case) which means it was probably better taken care of, and less likely to need aggressive cleaning, and more likely to work without needing any repairs. You can also wait to find one local that you can pick up in person (to save shipping cost and/or potential damage) or which will be cheaper to ship from nearby.

Without any experience, you might try looking at Just My Typewriter's Typewriter 101 series on YouTube: https://www.youtube.com/playlist?list=PLJtHauPh529XYHI5QNj5w9PUdi89pOXsS She covers most of the basics there.

Cleaning a machine isn't horribly difficult and can be done pretty cheaply ($20 or less for some paint thinner/isopropyl and a small toothbrush), but if you need it or get a machine that needs some repair work, try https://site.xavier.edu/polt/typewriters/tw-repair.html.

If you're in an area with lots of yard sales, try shopping around and see if you find something interesting. It's at these that you'll have a potential chance of finding more collectible machines for pennies on the dollar and it'll also give you the chance to put your hands on machines to test them out to make sure they work.

Good luck! 🎄

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reply to u/strawberystegosaurus at https://old.reddit.com/r/typewriters/comments/1g5rgi4/typewriter_for_christmas_please_help/

He is the only noble gas with 2 valence e and not 8

It’s easier for Non metals to gain electrons (become anions) to make the shell full aka octet rule

anyone knows what this means？

no entiendo por que Esposito hace eso, el hecho que en el personalismo no se tenga en cuenta el cuerpo, la verdad derrumba esta crítica de Esposito, la persona es cuerpo, pero no se reduce al cuerpo únicamente

I've actually seen lots of videos where the AI is biased towards one political candidate versus another, you can ask specific questions like "Why should I vote for Trump/Kamala" and you may receive biased answers

This would be super interesting, AI would be able to pry into what they may have believed about certain topics just solely based on their works and then be the person that wrote them.

I've only ever really used AI for research, this is an incredible approach to AI. Asking for help with specific problems or for help when improving an essay is a great idea.

In my experience with AI, I have never encountered an error.

It's interesting that they knock out the pros and cons of AI immediately in the beginning of the article. An approach I have not seen before.

This is incredibly interesting! I have never heard of Theseus before. It is super interesting to learn that AI has been here since before the internet.

AI-native social media doesn't seem that far away tho

how human-in-the-loop a tool is will also be a factor. IC humans will do more QA; higher-level humans will do more orchestration

bearish on prosumer AI?

should generational products always aim for this (i.e. FB and Google approach of doing work for people) instead of augmenting people's abilities?

improve the backend tech instead of simplifying the UX

key to young people utilizing tech in more interesting and innovative ways, I think, is that they simply have more free time to explore the product. adults are always busy and in a rush

they just needed access to a bicycle

sort of like a tour guide for translating biz reqs -> tech

the equivalent for Copilot now would be that's for people who don't currently code

VisiCalc gives people who couldn't do something before a step function change bc now they CAN do it; Copilot is a marginal improvement that relies on there being an existing behavior

sweet spot of bicycle-riding: it's what one augmented person can produce

what 10x more ambitious thing does the technological shift enable?

This is very surprising to hear. I don't think any of my teachers from HIgh School ever used AI and if they did, they didn't tell us or show us in any way.

While this is helpful, AI doesn't really think for itself, it sounds like thinking. I think AI is better used as a tool for understanding other topics better.

why not?

This shows that the creators of AI, OpenAI in particular is taking special attention to how the education industry has been affected and is offering a tool to support Higher Education.

I also appreciate the origin and definition of AI being present before we get into the meat of the article.

I like that they give us a clear reason to trust them and it really builds their ethos and credibility.

I would move this section up much higher on the page right underneath the kind words section.

I would also revamp this section entirely because the lessons are already released (I assume) and ready to watch as soon as they join. So I would say something like: Access additional short sessons from each of the guest artists as well as the othe amazing benefits waiting for you inside the membership.

And then I'd list the sessions as well as the other top-line inclusions of your membership in bullet point format.

Keep growing with your FREE month inside the Roben's Nest

and receive ONE MONTH FREE inside the Roben's Nest, the membership .... (valued at $24).

This exlusive launch offer and pricetag disappear when the timer strikes zero

and ongoing

I would make this the heading

virtual summit

I'd change the CTA here too... DOWNLOAD THE DIGITAL VERSION

I'm curious... are you not able to link to the print version or do you ask them to sign up so you capture their email addresses? If it's a conscious decision to make them sign up, I'd reword the button CTA slightly. GET YOUR EXCLUSIVE LINK TO ORDER... and then the CTA in the pop-up box would be SEND ME THE EXCLUSIVE LINK TO ORDER

I wonder whether you can include a photo of yourself with the print version in the header so there's a warm smile as soon as they land on the page.

Les quartiers sont rénovés. Ils ont une nouvelle image grâce à l'art de rue.*

L'art de rue attire les touristes. L'art de rue a une bonne position à Paris.

Cet article est sur l'art de rue à Paris. Il parle aux parisiens curieux. Il parle aussi aux visiteurs d'autres pays. Cet art aide le tourisme à Paris.

J'aime le titre de cet article

Can you add a little more detail around how many hours it includes and the outcome of the day?

I would move this up so it's in line with the inclusions in the column on the left. And bold the VIP day inclusion on the left...

I would centre this

This font seems different to the headings above?

Can you centre this section on the page and bold the first sentence?

Can you move this text next to the photo of your with your medal at all?

Could you structure this section as 4 columns at all? I think it would make it look cleaner.

I feel like there's something missing over the header image?

!!!!

Content specific NCC are neuronal populations which are active when specific "contents of experience" are perceived or thought about, and are inactive otherwise. For example, if the NCC for experiencing the contents of a face are stimulated

NCC can refer to content-specific NCC or full NCC.

Disorder of consciousness in which patients retain autonomic reflexes and the ability to spontaneously open their eyes despite being completely unresponsive in every other way.

An area of the posterior cerebral cortex spanning the temporal, parietal, and occipital lobes. There is a strong possibility that it is the location of the full and content specific NCC. Content specific NCC are populations of neurons associated with specific perceptual experiences (e.g., facial recognition).

An experimental model in which groups that provide subjective reports of what they perceive (a presented stimulus) are compared with groups that do not provide such reports. Instead, we attempt to determine what the latter group is perceiving via physiological measures (neuroimaging technology). This method allows the NCC to be distinguished from activity which relates to them.

In behavioral paradigms, consciousness is evaluated using verbal reports or physical responses indicating the perception of a presented stimulus. The accuracy of these reports/responses is unreliable when said stimuli are dubiously perceptible, because it is hard to tell whether the subject actually perceived the stimulus or simply guessed its position correctly. Forced-choice procedures can determine the subject's objective awareness of the stimulus by minimizing their subjective bias.

This is a continuation of Elliot showing his sympathy for Bonnie after realizing she was a victim of sexual assault. A great way of showing sympathy is to simply be in a space that brings the other comfort and just sitting with them in it.

This is the basic way of showing sympathy for someone but Elliott continues on by giving Bonnie a moment of comfort.

I think this is too much in the header section. I would remove this section entirely.

As for the button copy, I'd remove Register Here!

I would beef these up in a separate 'this is what you can expect' section underneath your about section.

Some of these ideas would be great to the 'right now' section further up on the page

I'd try to condense your about section to one visual section on the page. So possibly see how you can move this bit into your 'main' about section

BB ARt Journal Masterclass will help you...

I'd then follow it with a paragraph that addresses the right now and how they're currently approaching their art. The frustration of doubting themselves, not knowing where to start, lacking feedback so they know how they can improve, etc...

And then I would introduce your BB Art Journals Masterclass as the solution in the next section.

I would lead with what's possibe here... the dream future!

What if you activated your full creative potential and said goodbye to overwhelm?

Imagine what it would feel like to eliminate the sheer impossible obsticles that have been holidng you back from creating? And you not only started creating consistently, but you could watch your skills grow week by week?

And you could trust your artistic intuition?

Someting like this...

This also perfectly lends itself to a clumn-style approach

I would definitely turn this into a 3-colum section with a matching icon/graphic for each

I would move this section right underneath the header

Find confidence through creativity in this 12-week art adventure, and FINALLY embrace your unique style

A nice little fact about the top 3 country

I like how it is also in Spanish

This is the website bringing to a springer link. It's a research website

Not too recent but in a span of 5 years

Obinna is a researcher, her research are mainly about immigration, social policy and challenges faced by minorities.

Java promotes smaller data types (i.e., byte, short, and char) to int for arithmetic operations to prevent overflow issues. This process is called type promotion.

I think Ellis' ideology regarding a link between racial questions and the question of "sex" is largely a problem with interracial romantic and sexual relationships, and perhaps the morality of sex for pleasure rather than only procreation. Interracial relationships are often scrutinized under the idea of a black man being sexually violent towards a white women, therefore creating prejudice against any variation of interracial coupling. Protesting sex for pleasure is often connected with conservative ideologies that control women's bodies under a "pro-life" facade. This protest attempts to create an option of have more children or remain abstinent, however this is an unrealistic expectation and leads to the deaths of many women who received unsafe abortions.

The United States classifying bodies as "black," "white," "heterosexual," or "homosexual" is a tactic to create a social other and control bodies. Similarly this is seen when classifying bodies as "male" and "female" and the control the government instills over feminine reproductive rights.

Has sexuality become seen as a social construct in the same way gender is because it is being classified as an "invention"? Could it therefore be argued that certain societies with different conventions have no, less, or more sexualities and genders than modern society?

Can I have binary files in my notes??

It seems that you're able to make notes on commits. Since a commit can be referenced by a tag, or branch, you can make notes on those, too -- kind of.

I have wondered if git's notes function would allow one to keep some kind of issue management system within the repository itself.

Social class due to morphological and physiological differences

Sacrifice life in order to help closely related oheims to survive

Unable to produce children

High school Bio then were two types of survived ship curve to increase population

make (something, typically an undesirable situation or an unfounded belief) continue indefinitely.

Ignore this highlight

This quote from Duncan shows the frustration that he and other British had towards the colonists for questioning anything that England does. They viewed themselves as completely superior, so anything out of line triggered them. Cora's response reminds him of the hardships the colonist experienced to settle there as well as what it costed them. This is a good example of the tensions that started to arise between the colonists and the British; where they are starting to form their own identity without the authority of England.

• This wasn't related to any readings for this movie, but signs of colonists revolting was brought up during the lecture that week.

In these couple lines, Webb is basically saying that the King would accept the colonists defense against any "savage" attack, especially if they were brought on by the French. This portrays the small conflicts that were going on between the French and the British where the colonists would have to pay. Comparing this to readings of that time, these events seem historically accurate as there were raids carried out by Native Americans on their former lands.

• Calloway, First Peoples, 168-169

This matched with what actually happened to Fort William Henry, where the French put constant pressure on the fort. The French knew that the British had to give in eventually, and when supplies ran low they did.

• Greig Santos-Buch. 2024. “Understanding the Siege and Battle at Fort William Henry.” May 15, 2024.

Gifts and trade were one of, if not the most popular ways of diplomacy between the natives and the British/French. This quote by Hawkeye describes that, but instead he talks about how the use of these gifts manipulate tribes into giving up their cultural values. This portrays colonialism in one of it's worst forms, where it tears apart tribes and lands.

• Calloway, World Turned Upside Down, 140

Magua's line's here represents the culture disconnect from the Natives and the British. One of these biggest contrasts are the gender roles, where women in British culture relied on men more than in Native cultures. This also speaks to the very negative view that lots of tribes had on the British.

• “The British Era.” n.d. Minnesota Historical Society.

The romantic and deeply mythological lines here serve to help understand the historical context they were in. Lots of captures by Indians were treated harshly, but some, especially women and children, weren't as they might have been adopted into the tribe's culture. Here is a specific saying that is only known in Hawkeye's culture, a sign that he is not only opening up to Cora but maybe wants her to learn his culture. Also, it is believed that the Natives had a deep respect for nature and that the elements were connected with them spiritually.

• Calloway, First Peoples, 169-170

Obviously this is a TV show, it this does happen to so many women with families. I am very strong willed and would not put up with being so submissive or subtle. People and children need to be aware of people’s feelings. And as a parent and wife you need to be strong enough emotionally to communicate that.

Very true. At the end of the day we are all human. Sometimes we just have to be more aware of how we interact with other cultures, because we would not want to come across as rude or insensitive.

This is very true! Especially when going to big cities it’s easy to be disgusted at the homelessness and drug use, it’s hard to not make faces or want to help. In bigger cities it’s more common to hide your facial expressions and just look at the ground instead of a more blunt approach.

This is a very interesting example of how much power the internet itself holds. The fact that K-pop fans managed to crash an entire software is quite amazing. Similar to the fact that 4chan users arranged a whole rendezvous for single men just to laugh at them, but for a greater cause. K-pop has only grown since 2020, which may very well eventually raise the question of what other tricks they have up their sleeve.

Another funny instance of this I thought of were the sugarfree Haribo Gummy Bears. The sweetener used in the bears were actually laxatives, and so online users went ahead and posted all types of wild parody stories of them eating the bears and suffering immense digestive pain from destroying the toilet to going to the hospital. While parody reviews definitely have a humor factor, I also think that they have the potential to make or break a product based on the overall perceived notion of the parody reviews collective.

I feel like people would pay more attention to what is taught in schools if they read this sentence. Not only because they would learn that Hitler got his idea for concentration camps from the United States but because of the statement, "Ironically, Adolf Hitler displayed more knowledge of how we treated Native Americans than American high schoolers today who rely on their textbooks." This is a scary thought, In a sense, Loewen is highlighting that a terrible man in history, who is not an American, knows more about American history than kids sitting in a classroom in the United States. I feel like this statement alone sums up just how much the education system keeps from us. It kind of makes me mad to think that horrible dictator knew more about my country than I do. I feel like more people should feel this way and make sure a change happens for future generations.

This is a great start to how we should think about the information that is exposed to students. The main problem that we see from this book is that the education system does not think students are capable of forming their own opinions about history. I feel like that is why a lot of the history we learn in high school is 'watered down," so to speak. I believe the education system can make up for its mistakes by doing its research. The problem is that we do not know if everyone who is in charge of what is required for students to learn understands what information is being left out. They simply could be going with what they know. The other problem is that, sadly, there are people in charge of the education system who know exactly what FACTS they are withholding. Hopefully, deceiving of what truly happened in our country's history will change shortly.

This statement shocked me. While we didn't go into deep detail about the Louisiana Purchase, it was emphasized that the United States bought the land from France. It is disappointing that this piece of history was withheld from my education. Loewen is correct when discussing how the Indians were portrayed in our education. All my life, I thought the stories of Native Americans not understanding land ownership were true. Because, sadly, those stories made sense. Native Americans were painted as people who just lived by the land and had no rule of law. However, Loewen gives us many examples of how the education system failed us by not debunking that statement,

First Instance: * Thesiger J. suggested sellers' terms "shall prevail" bound future dealings, implying buyer agreement. * Rejected on appeal to prevent unfair imposition of terms by offerors. Second Suggestion (Professor Guest): Divergence in reply leads to a counter-offer; acceptance could be implied from silence. * Concerns raised by Bridge L.J. and Lord Denning about the implications of this approach. Example Concern: * If a counter-offer significantly undercuts the original offer (e.g., £72,000 vs. £60,000), sellers may not expect a response, aligning with traditional commercial expectations.

A definite and seasonable expression of acceptance or a written confirmation which is sent within a reasonable time operates as an acceptance even though it states terms additional to or different from those offered or agreed upon, unless acceptance is expressly made conditional on assent to the additional or different terms

1. Timely acceptance valid even with different terms unless: Acceptance conditions on agreeing to additional terms.
2. Additional terms: Considered proposals for the contract. Become part of the contract between merchants unless: Offer limits acceptance to original terms. Terms materially alter the contract. Objection is given promptly.

formation of accpt

This quote shows how the executions reinforced the oppressive power supporting slavery. Also, the idea that racism was further developed due to the treat of rebellion.

Test

The passage effectively highlights the similarities in Hoover and Al Smith's platforms but could expand on why religion and Prohibition became focal points of the 1928 campaign. Adding more context about Hoover’s rise as Coolidge’s natural successor and his public service background would strengthen the analysis.

Despite underlying issues in both industrial and agricultural sectors, many Americans in 1929 and 1930 remained optimistic about a quick economic recovery. President Herbert Hoover reinforced this belief by confidently declaring in 1930 that “the depression is over,” underestimating the severity of the crisis.

Despite efforts by Populists and Progressives, economic inequality widened in the early 20th century, with wealth concentrating among the rich. Conservative policies in the 1920s, including tax cuts and deregulation, fueled this divide, benefiting the wealthiest Americans far more than the general population.

The Stock Market Crash was important because it marked the sudden collapse of an inflated economy fueled by speculation, leading to widespread financial panic. This crash triggered a chain reaction of bank failures, mass unemployment, and economic hardship that defined the Great Depression in the U.S. and globally.

Though the romance between Pocahontas and John Smith never happened, Pocahontas might have influenced the fate of John Smith. According to the Park Service, a theory is Pocahontas placed her head upon his when the Chief was about to bash his head open, but this has been debated for years.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

“Pocahontas: Her Life and Legend.” National Park Service, September 4, 2022. https://www.nps.gov/jame/learn/historyculture/pocahontas-her-life-and-legend.htm

This line shows the English's lack of empathy for anyone or anything due to the selfish drive to get rich. They believed that because they came there they are now the rulers of the land and the Natives are just uncivilized savages, which is far from the truth.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

When John asked Pocahontas about the gold, as shown in these lines, she thought he was talking about corn. Native Americans did not see gold as something of value, they valued resources like crops and animal skins. They lived by what they needed, not what they wanted. The British were after what they wanted, a material that was given value, not inherently valuable.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

These lines further explore the conceptions the British people had about Natives. The British believed that their way of life was superior. Though they took it upon themselves to rob and fight the natives because of the natives' skilled way of the land. They had captivated the land, and the British just replied on trade, occasionally, and taking it from them. Yet, they thought their way of life was more "civilized".

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

This excerpt of the script shows the context in which Natives have knowledge of the land and how to benefit from it. Unlike the Natives, the British did not understand how to captivate the potentials of the land. They assumed the land would already be rich in nutrients, and didn't prepare to be laborers.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

This excerpt from the script portrays the English view on Native Americans during this time. They referred to them as "savages" and treated them with no respect. Native Americans were seen as an obstacle to the "get rich quick" goal they were pursuing.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

This excerpt of the script further proves the point that the most important goal the British had was to get rich and claim their riches in Britain. Colonizing wasn't necessarily a goal at first.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

When the English arrived, the Native Americans were already fighting amongst themselves. They understood the land and its advantages and disadvantages, unlike the British.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

The filmmakers tried to portray the ideals the British had when setting sail to the Americas. The Script indicates the Virginia Company believed the land would be filled with gold and rich with valuable resources, yet this was far from the truth.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

'scrap' tnv amd replace them by 'reasonable implementation'

This sentence that I highlighted the growth of colonial settlements into powerful self sustaining places. The statement "warring against Native Americans" refers to the constant conflict between European settlers and the indigenous people which were often fought over land and resources.This reflect the mindset of the colonist who viewed the land as theirs regardless of native being there prior

The sentence that I highlighted the growth of colonial settlements into powerful self-sustaining places. The statement "warring against the Native American" refers to the constant conflict between the European settlers and the indigenous people which often fought over land and resources. This reflects the mindset of colonist who viewed the land as their own prior to the Native American being there before them. The phrase 'subduing internal upheaval' probably refers to both the political and social struggles with the colonies.

The mentality of “I hit the game winning shot because I’m the best” vs “I missed the game winning shot because you didn’t get me the ball fast enough” Fascinating to see these examples from life be put into a structured explanation.have a coworker who, if there is a mistake, will always be the last to get back to the office and then proceed put the blame on someone else. If the job is completed on time, looks good and mistake free he will be the first one in the office reporting to the boss how good he did on the job.

This fundamental attribution error seems to be a rabbit hole our society is falling down. I had always referred to it as victim culture. We as a society are unable to see how our external attribution to a situation plays a role in the outcomes in life. Everything bad that happens to us is because somebody else is bad and out to get me. It is much easier to blame someone else, for reasons your assuming, then it is to realize the mistakes that were made.

Producer closes their channels. Consumers should exit when the channel they're polling on is closed.

Receive on closed channel's fine.

Since I was younger I’ve known that we have beliefs that are instilled dingo us, knowingly and unknowingly. The part that is always difficult for me is how do I notice them, and how do I change them. One of the Schemata I have realized is the way I perceive money. I have always felt like I never had enough, and it was going to run out. However, after 10 years of working, I have never gone without, been homeless or starved. I recognize the schema now I am focused on changing that database.

There is something similar in the psychology realm called Baader-Meinhoff. This is the phenomenon where you buy something, say a new silver Toyota Camery. All of a sudden it feels like every car you are seeing on the roads is a silver Toyota Camery. This happened to me when I bought my new truck, it made me think I made a good decision because of how many other people had the same truck. I wasn’t looking for the truck but it seemed like it was the only vehicle on the road that ever caught my attention.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

The Notch signaling pathway plays an important role in many developmental and disease processes. Although well-studied there remain many puzzling aspects. One is the fact that as well as activating the receptor through trans-activation, the transmembrane ligands can interact with receptors present in the same cell. These cis-interactions are usually inhibitory, but in some cases, as in the assays used here, they may also be activating. With a total of 6 ligands and 4 receptors, there is potentially a wide array of possible outcomes when different combinations are co-expressed in vivo. Here the authors set out to make a systematic analysis of the qualitative and quantitative differences in the signaling output from different receptor-ligand combinations, generating sets of "signaling" (ligand expressing) and "receiving" (receptor +/- ligand expressing cells).

The readout of pathway activity is transcriptional, relying on the fusion of GAL4 in the intracellular part of the receptor. Positive ligand interactions result in the proteolytic release of Gal4 that turns on the expression of H2B-citrine. As an indicator of ligand and receptor expression levels, they are linked via TA to H2B mCherry and H2B mTurq expression respectively. The authors also manipulate the expression of the glycosyltransferase Lunatic-Fringe (LFng) that modifies the EGF repeats in the extracellular domains impacting their interactions. The testing of multiple ligand-receptor combinations at varying expression levels is a tour de force, with over 50 stable cell lines generated, and yields valuable insights although as a whole, the results are quite complex.

Strengths:

Taking a reductionist approach to testing systematically differences in the signaling strength, binding strength, and cis-interactions from the different ligands in the context of the Notch1 and Notch 2 receptors (they justify well the choice of players to test via this approach) produces a baseline understanding of the different properties and leads to some unexpected and interesting findings. Notably:

-                Jag1 ligand expressing cells failed to activate Notch1 receptor although were capable of activating Notch2. Conversely, Jag2 cells elicited the strongest activation of both receptors. The results with

Jag1 are surprising also because it exhibits some of the strongest binding to plate-bound ligands. The failure to activate Notch1 has major functional significance and it will be important in the future to understand the mechanistic basis.

-                Jagged ligands have the strongest cis-inhibitory effects and the receptors differ in their sensitivity to cis-inhibition by Dll ligands. These observations are in keeping with earlier in vivo and cell culture studies. More referencing of those would better place the work in context but it nicely supports and extends previous studies that were conducted in different ways.

-                Responses to most trans-activating ligands showed a degree of ultrasensitivity but this was not the case for cis-interactions where effects were more linear. This has implications for the way the two mechanisms operate and for how the signaling levels will be impacted by ligand expression levels.

-                Qualitatively similar results are obtained in a second cell line, suggesting they reflect fundamental properties of the ligands/receptors.

We appreciate the positive and constructive feedback.

Weaknesses:

One weakness is that the methods used to quantify the expression of ligands and receptors rely on the co-translation of tagged nuclear H2B proteins. These may not accurately capture surface levels/correctly modified transmembrane proteins. In general, the multiple conditions tested partly compensate for the concerns - for example, as Jag1 cells do activate Notch2 even if they do not activate Notch1 some Jag1 must be getting to the surface. But even with Notch2, Jag1 activities are on the lower side, making it important to clarify, especially given the different outcomes with the plated ligands. Similarly, is the fact that all ligands "signalled strongest to Notch2" an inherent property or due to differences in surface levels of Notch 2 compared to Notch1? The results would be considerably strengthened by calibration of the ligand/receptor levels (and ideally their sub-cellular localizations). Assessing the membrane protein levels would be relatively straightforward to perform on some of the basic conditions because their ligand constructs contain Flag tags, making it plausible to relate surface protein to H2B, and there are antibodies available for Notch1 and Notch2.

We agree that mCherry fluorescence does not provide a direct readout of active surface ligand levels. As the reviewer points out, the ability of Jag1 to activate Notch2 demonstrates that expressed Jag1 is competent for signaling. Further, in some cases, Jag1-Notch2 activation can be comparable to Dll1-Notch2 activation (Figure 2A). Following the reviewer’s suggestion, we performed a Western blot for multiple expression levels for each of three surface ligands (Dll1, Dll4, Jag1) (Figure 2—figure supplement 2). This blot revealed a signal for surface expression of Jag1. Interpretation is complicated by the expected dependence of the efficiency of surface protein purification on the number of primary amines in the protein, which varies among these ligands, and qualitatively correlates with the staining intensity. While this makes quantitative interpretation difficult, this result further supports the notion that Jag1 is present on the cell surface. Finally, we note that high signaling activity need not, in general, directly correlate with surface expression levels. In fact, one study showed an example in which increased ligand activity occurred with decreased basal ligand surface levels (Antfolk et al., 2017). While one would ideally like to know all parameters of the system, including surface protein levels, rates of recycling, etc. the perspective taken here is that the net effect of these many post-translational processing steps can be subsumed into the overall relationship between the expression of the protein (which, in our case, is read out by the co-translational reporter) and its activity, which is relevant for the behavior of developmental circuits, among other systems. To address this comment, we now explicitly mention the limitation of mCherry as a proxy for surface protein, and add a reference to previous work highlighting the relationship between surface levels and ligand activity.

In terms of the dependence of signaling on Notch levels, the metric of signaling activity used here is explicitly normalized by the mTurquoise co-translational reporter of Notch expression to account for differences in receptor expression across receiver clones. We have added a new figure to show the variation in expression (Figure 1—figure supplement 1A) and to demonstrate this normalization (Figure 1—figure supplement 5). Having said that, as the reviewer correctly points out, we cannot directly address the dependence on surface receptor levels with mTurquoise alone. To address this comment, we have added a figure that shows cotranslational and surface receptor expression for a subset of our receiver clones (Figure 1—figure supplement 1B). Although antibody binding strengths may vary, it appears unlikely that higher surface levels could explain most ligands’ preferential activation of Notch2 over Notch1, since Notch2 levels were lower than Notch1 levels in both surface expression and cotranslational expression.

Cis-activation as a mode of signaling has only emerged from these synthetic cell culture assays raising questions about its physiological relevance. Cis-activation is only seen at the higher ligand (Dll1, Dll4) levels, how physiological are the expression levels of the ligands/receptors in these assays? Is it likely that this would make a major contribution in vivo? Is it possible that the cells convert themselves into "signaling" and "receiving" sub-populations within the culture by post-translational mechanism? Again some analysis of the ligand/receptors in the cultures would be a valuable addition to show whether or not there are major heterogeneities.

The cis-activation results in this paper are, as the reviewer points out, conducted in synthetic cell culture assays. Cis-activation is observed across a large dynamic range of ligand expression, possibly including non-physiologically high levels. However, our previous work (Nandagopal et al, eLife 2019) showed that cis-activation does not require over-expression, as it occurred in unmodified Caco-2 and NMuMG cells with their endogenous ligand and receptor expression levels. As shown here in Figure 4B, cis-activation for Notch2 increases monotonically and is substantial even at intermediate ligand concentrations. In other cases, cis-activation is maximal at intermediate concentrations. We agree that the in vivo role remains unclear, and is difficult to determine due to the typical close contacts among cells in tissues. Therefore, these assays do not speak to in vivo relevance. Note that we can, however, rule out the possibility of trans signaling between well-mixed cell populations at these densities (Figure 4A).

It is hard to appreciate how much cell-to-cell variability in the "output" there is. For example, low "outputs" could arise from fewer cells becoming activated or from all cells being activated less. As presented, only the latter is considered. That may be already evident in their data, but not easy for the reader to distinguish from the way they are presented. For example, in many of the graphs, data have been processed through multiple steps of normalization. Some discussion/consideration of this point is needed.

We agree that in different experiments changes in a mean response can reflect changes in fraction of activated cells, or level of activation or some combination of both. In this work, most assays were conducted by flow cytometry, which provides a full distribution of cellular responses. We provided distributions for some experiments in the supplementary figures (i.e., Figure 4—figure supplement 1, and Figure 5—figure supplement 4). The sheer number of experiments and samples prevents us from displaying all underlying histograms. Therefore, we have provided all flow data sets in an extensive archive that is publicly available on data.caltech.edu (https://doi.org/10.22002/gjjkn-wrj28).

Impact:

Overall, cataloging the outcomes from the different ligand-receptor combinations, both in cis and trans, yields a valuable baseline for those investigating their functional roles in different contexts. There is still a long way to go before it will be possible to make a predictive model for outcomes based on expression levels, but this work gives an idea about the landscape and the complexities. This is especially important now that signaling relationships are frequently hypothesized based on single-cell transcriptomic data. The results presented here demonstrate that the relationships are not straightforward when multiple players are involved.

We appreciate this concise impact summary, and agree with its conclusions.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors extend their previous studies on trans-activation, cis-inhibition (PMID: 25255098), and cis-activation (PMID: 30628888) of the Notch pathway. Here they create a large number of cell lines using CHO-K1 and C2C12 cells expressing either Notch1-Gal4 or Notch2-Gal4 receptors which express a fluorescent protein upon receptor activation (receiver cells). For cis-inhibition and cis-activation assays, these cells were engineered to express one of the four canonical Notch ligands (Dll1, Dll4, Jag1, Jag2) under tetracycline control. Some of the receiver cells were also transfected with a Lunatic fringe (Lfng) plasmid to produce cells with a range of Lfng expression levels. Sender cells expressing all of the canonical ligands were also produced. Cells were mixed in a variety of co-culture assays to highlight trans-activation, cis-activation, and cis-inhibition. All four ligands were able to trans-activate Notch1 and Notch 2, except Jag1 did not transactivate Notch1. Lfng enhanced trans-activation of both Notch receptors by Dll1 and Dll2, and inhibited Notch1 activation by Jag2 and Notch2 activation by both Jag 1 and Jag2. Cis-expression of all four ligands was predominantly inhibitory, but Dll1 and Dll4 showed strong cis-activation of Notch2. Interestingly, cis-ligands preferentially inhibited trans-activation by the same ligand, with varying effects on other trans-ligands.

Strengths:

This represents the most comprehensive and rigorous analysis of the effects of canonical ligands on cis- and trans-activation, and cis-inhibition, of Notch1 and Notch2 in the presence or absence of Lfng so far. Studying cis-inhibition and cis-activation is difficult in vivo due to the presence of multiple Notch ligands and receptors (and Fringes) that often occur in single cells. The methods described here are a step towards generating cells expressing more complex arrays of ligands, receptors, and Fringes to better mimic in vivo effects on Notch function.

In addition, the fact that their transactivation results with most ligands on Notch1 and 2 in the presence or absence of Lfng were largely consistent with previous publications provides confidence that the author's assays are working properly.

We appreciate the thoughtful comments and feedback.

Weaknesses:

It was unusual that the engineered CHO cells expressing Notch1-Gal4 were not activated at all by co-culture with Jag1-expressing CHO cells. Many previous reports have shown that Jag1 can activate Notch1 in co-culture assays, including when Notch1 was expressed in CHO cells. Interestingly, when the authors used Jag1-Fc in a plate coating assay, it did activate Notch1 and could be inhibited by the expression of Lfng.

In our assays, we do in fact also see some signaling of Jag1 to Notch1, especially when dLfng is coexpressed (Figure 2—figure supplement 4, formerly Figure 2—figure supplement 3). While these levels are lower than those observed for other ligand-receptor combinations, they are significantly elevated compared to baseline. In specific natural contexts, it will be important to determine whether the weak but non-zero Jag1-Notch1 signaling acts negatively to suppress signaling from other ligands, or provides weak but potentially functionally important levels of signaling. Evidence for both modes exists in the literature. To address this, we have expanded the discussion of Jag1-Notch1 signaling and added references to other work on Jag1-Notch1 signaling to the Discussion section.

The cell surface level of the ligands was determined by flow cytometry of a co-translated fluorescent protein. Some calibration of the actual cell surface levels with the fluorescent protein would strengthen the results.

This issue was also raised by Reviewers #1 and #3. Please see responses to Reviewer #1, above.

Reviewer #3 (Public Review):

Summary:

This manuscript reports a comprehensive analysis of Notch-Delta/Jagged signaling inclusive of the human Notch1 and Notch2 receptors and DLL1, DLL4, JAG1, and JAG2 ligands. Measurements

encompassed signaling activity for ligand trans-activation, cis-activation, cis-inhibition, and activity modulation by Lfng. The most striking observations of the study are that JAG1 has no detectable activity as a Notch1 ligand when presented on a cell (though it does have activity when immobilized on a surface), even though it is an effective cis-inhibitor of Notch1 signaling by other ligands, and that DLL1 and DLL4 exhibit cis-activating activity for Notch1 and especially for Notch2. Notwithstanding the artificiality of the system and some of its shortcomings, the results should nevertheless be a valuable resource for the Notch signaling community.

Strengths:

(1)  The work is systematic and comprehensive, addressing questions that are of importance to the community of researchers investigating mammalian Notch proteins, their activation by ligands, and the modulation of ligand activity by LFng.

(2)  A quantitative and thorough analysis of the data is presented.

Weaknesses:

(1) The manuscript is primarily descriptive and does not delve into the underlying, mechanistic origin or source of the different ligand activities.

We agree that the goals of this paper were largely to discover the range of signaling modes that occur. A mechanistic analysis would be beyond the scope of this work, but we agree it is an important next step.

(2) The amount of ligand or receptor expressed is inferred from the flow cytometry signal of a co-translated fluorescent protein-histone fusion, and is not directly measured. The work would be more compelling if the amount of ligand present on the cell surface were directly measured with anti-ligand antibodies, rather than inferred from measurements of the fluorescent protein-histone fusion.

This issue was also raised by Reviewers #1 and #2. Please see responses to Reviewer #1, above.

(3) It would be helpful to see plots of the raw activity data before transformation and normalization, because the plots present data after several processing steps, and it is not clear how the processed data relate to the original values determined in each measurement.

We included examples showing how raw data is processed in Figure 4—figure supplement 1 and Figure 5—figure supplement 4. The sheer number of experiments precludes including similar figures for all data sets. However, all raw and processed data and data analysis code is publicly available at (https://doi.org/10.22002/gjjkn-wrj28).

(4) The authors use sparse plating of engineered cells with parental (no ligand or receptor-expressing cell to measure cis activation). However, the cells divide within the cultured period of 22-24 h and can potentially trans-activate each other.

If measured cis-activation signal arises solely from trans-activation, then the measured cis-activation signal per cell should increase with cell density, since trans-activation per cell does depend on cell density (Figure 4A). However, for the strongest cis-activators (Dll1- and Dll4-Notch2), signaling magnitude is similar when these cells are cultured sparsely or at confluence, which would otherwise allow efficient trans signaling (Figure 5A). Thus, for Dll1- and Dll4-Notch2 receivers, total signaling strength per cell depends little or not at all on the opportunity to signal intercellularly. Moreover, cis-activation signal for the Dll1- and Dll4-Notch2 combinations exceeded the maximum trans-signaling levels we could achieve for the same receivers when cis-ligand was suppressed (Figure 4B). These results argue that cis interactions dominate signaling in this context. However, we have not ruled out the possibility that trans-signaling between sister cells after division contributes to the comparatively weak cis-activation observed for Notch1 receivers.

Reviewer #1 (Recommendations For The Authors):

As outlined in the public review, there is a question of whether the nuclear H2B accurately reflects the surface levels of the transmembrane proteins (ligand and receptor). Clearly, it would not be feasible to check levels in all of the experimental conditions, but some baseline conditions should be analyzed.

We addressed this above.

Reviewer #2 (Recommendations For The Authors):

(1)  As mentioned above, it was unusual that Jag1 did not activate Notch1 in co-culture assays, but did activate Notch1 in plate-coating assays. The authors should add some text to the Discussion to explain why they think this is happening in their engineered cells. One possibility is that the CHO cells express Manic fringe (Mfng) which is known to reduce Jag1-Notch1 activation. Data for Mfng levels in CHO cells were not included in Supplemental Table 2. Knocking down all three Fringes in CHO cells might increase Jag1-Notch1 activation.

This is already addressed in a sentence in the results: “Strikingly, while Jag1 sender cells failed to activate Notch1 receivers above background (Figure 2D), plate-bound Jag1-ext-Fc activated Notch1 only ~3-fold less efficiently than it activated Notch2 (Figure 3B-D). This suggests that the natural endocytic activation mechanism, or potential differences in tertiary structure between the expressed and recombinant Jag1 extracellular domains, could play roles in preventing Jag1-Notch1 signaling in coculture.” Regarding the point about Mfng, we added a note to Supplementary Table about other CHO-K1 expression data.

(2) Figure 1-supplemental figure 1: Both the Notch1-Jag1 and Notch1-Jag2 cells show high expression of Jag1 in low 4epi, but any higher concentration reduces to control levels. How much of a problem is this for interpreting your data?

This was not the ideal behavior, but by binning cells by co-translational reporters for ligand expression, we were able to obtain enough cells in intermediate bins. (Note: Figure 1—figure supplement 1 is now Figure 1—figure supplement 2.)

(3)  Figure 1C legend: Are these stably-expressing cells or Tet-off cells? Please state in legend.

The figure legend has been updated.

(4)  Figure 1E: How long is the knockdown of Rfng and Lfng effective? Does it affect the expression of Lfng later?

siRNA effects generally last for at least 72-96 hours, so we do not anticipate this being an issue.

(5) Page 9: "Lfng significantly decreased trans-activation of both receptors by Jag1 (>2.5-fold)". If there is no Jag1-Notch1 activation, how can Lfng decrease trans-activation?

We added a note in the main text to clarify that while Jag1-Notch1 signaling is relatively low, it can still be detectably decreased.

(6) Figure 4A legend: Please define what "2.5k ea senders and Rec" means. In the text, it says "To focus on cis-interactions alone, we then cultured receiver cells at low density, amid an excess of wildtype CHO-K1 cells" (page 14).

This was clarified in the text.

(7)  Page 14: "By contrast, Notch2 was cis-activated by both Dll1 and Dll4, to levels exceeding those produced by trans-activation by high-Dll1 senders (Figure 4B, lower left)." Where is the trans-activation data? 4B, lower right?

We updated this reference in the main text.

(8)  Page 16: "For Notch2-Dll1 and Notch2-Dll4, single cell reporter activities correlated with cis-ligand expression, regardless of whether cells were pre-induced at a high or low culture density (Figure 4D)." It appears that Notch2-Dll1 has lower Notch activation at sparse culture than confluent.

We agree that the level signaling is lower in sparse compared to confluent on average. This is explained by the sensitivity of the Tet-OFF promoter to culture density (Figure 4—figure supplement 2). However, the key point of this experiment is the positive correlation, which is consistent with cis-activation, and inconsistent with the pre-generation of NEXT hypothesis diagrammed in Figure 4C, which would not be expected to produce such a correlation.

(9a) For the creation of the C2C12-Nkd cells: Has genomic sequencing been done to confirm editing of Notch2 and Jag1 loci?

We confirmed the knockdown but did not do genomic sequencing.

(9b) The gel in Figure 7-Supplement 1C is not adequate for showing loss of Jag1. It should be repeated.

In this case, we have only the single gel. We added a note in figure legend that no duplicate was performed.

(10) Figure 7A: Which Fringes are expressed in C2C12 cells? You should provide a rationale for knocking down just Rfng.

Figure 7—figure supplement 1A shows the levels of expression in C2C12. Note that Mfng is not highlighted because its levels were undetectable.

(11) Figure 7-Supplement 1D: This is confusing. Notch2 levels are not reduced in the left panel, and Notch1 and Notch2 levels are not reduced in the right panel?

C2C12-Nkd cells exhibit reduced levels of Notch1 and Notch3. This can be seen in Figure 7—figure supplement 1A. Panel D presents the results of additional siRNA knockdown, performed to prevent subsequent up-regulation of Notch1 and Notch3 during the assay. These knockdown results were variable, as shown. The Notch2 siRNA knockdown was not essential for these experiments, but performed despite very low levels of Notch2 to begin with. In the revision, we have added this note to the Methods.

Reviewer #3 (Recommendations For The Authors):

(1) The results section of the manuscript is very dense and difficult to follow, as are the figure legends.

We appreciate the criticism, and regret that it is not easier to read in its current form.

(2) The authors could emphasize areas of concordance with published results (where available) to place their artificial, engineered system into a better biological context. Are there any examples of studies in whole organisms where cis-activation plays a role?

We are not aware of examples of cis-activation in whole organisms at this point.

(3) How do the authors rationalize the different responses of Notch1 to cell-presented Jag1 as opposed to immobilized Jag1, where its signal strength is second in rank order on a molar basis?

This comment was addressed above in response to the first recommendation from Reviewer #2.

It is also difficult to understand Figure 2_—_figure Supplement 3B, in which it appears that Jag1 induces a Notch1 reporter response when LFng is knocked down (dLfng), and how those data relate to the inactive response to Jag1 shown in the main figures.

The issue here is a difference of normalization. Figure 2A in the main text is normalized to the sender expression level, i.e. relative signaling strength. By contrast, Figure 2—figure supplement 4B (previously Figure 2—figure supplement 3B) shows absolute signaling activity, which can appear higher because it does not normalize for ligand expression. For Jag1-Notch1 signaling in particular, substantial signaling required very high levels of Jag1. We have added a new figure to demonstrate these two types of normalization (Figure 2—figure supplement 1A).

See the Authr response image 1 below for a direct comparison of these two normalization modes using data from both Figure 2A and Figure 2—figure supplement 4B. Note how the Jag1-Notch1 signaling activities that are nonzero in the top plot go to zero in the bottom plot as a result of normalizing the values to ligand expression.

Author response image 1.

Comparison of normalization modes in Figure 2A and Figure 2—figure supplement 4B (formerly 3B).

Normalized trans-activation signaling activities for different ligand-receptor combinations (with dLfng only), either with further normalization to ligand expression (bottom row) or without further normalization (top row). Normalized signaling activity is defined as reporter activity (mCitrine, A.U.) divided by cotranslational receptor expression (mTurq2, A.U.), normalized to the strongest biological replicate-averaged signaling activity across all ligand-receptor-Lfng combinations in this experiment. Saturated data points, defined here as those with normalized signaling activity over 0.75 in both dLfng and Lfng conditions, were excluded. Colors indicate the identity of the trans-ligand expressed by cocultured sender cells. Error bars denote bootstrapped 95% confidence intervals (Methods), in this case sampled from the number of biological replicates given in the legend—n1 (for Notch1) or n2 (for Notch2). See Methods and Figure 2A caption for more details. Note that the only difference between this figure and the new Figure 2—figure supplement 1A is that this figure additionally includes the Jag1-high data from Figure 2—figure supplement 4B.

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