this memory is burned in his mind meaning it is there forever

He has PTSD, he is remembering to sounds of th explosiosns and people dying

he probably experienced a traumatic event during the Vietnam war that is related to this burning girl that he is talking about

he is heavily thinking about this girl

The cause of this sadness if due to a girl that he has lost.

could he possibly be having war flasbacks?

a girl stuck in the speakers thoughts

The author remembers the accident of the burning girl.

that a piece of land is burning

The screams are due to a burning image in the authors head.

He can picture her still crying from the pain he put her threw.

he is recalling the event, which happened years ago

Maybe he saw a girl crying when he was in the war and its been repeating in his mind.

Memorys of a girl hes known in the past

Possible remembering a memory of a girl he once loved or cared about.

The feelings for this girl are always inside his head throughout the whole day.

the person i used to be still makes them self known in my head

sounds like he is sad over a girl

he has a bad memory is his head

Right away he is talking about a girl. So the rest of the poem should tell me who and what this girl means to him.

SciScore for 10.1101/2021.05.14.21257059: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04318015</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Hydroxychloroquine Chemoprophylaxis in Healthcare Personnel …</td></tr></table>

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

fact

She says the skull she was looking for is unlike any other in the mass grave she was looking through. The eyes are described as holes, which creates a picture in my head of deep black holes as eyes in a decrepit skull.

I really appreciate the respect given to indigenous knowledge here.

The researchers could have gone much further in depth in describing it and the aspects of what they mean by cultural "safety". They've done a disservice here by downplaying widespread adoption. Why not? Why couldn't we accord the proper respect of traditions to actively help make these techniques more widespread? Shouldn't we be willing to do the actual work to accord respect and passing on of these knowledges?

Given my reading in the area, there seems to be an inordinate amount of (Western) "mysticism" attributed to these techniques (here and in the broader anthropology literature) rather than approaching them head-on from a more indigenous perspective. Naturally the difficult part is being trusted enough by tribal elders to be taught these methods to be able to pass them on. (Link this idea to Tim Ingold's first chapter of Anthropology: Why It Matters.)

All this being said, the general methods known from the West, could still be modified to facilitate in widespread adoption of those techniques we do know. Further work and refinement of them could continue apace while still maintaining the proper respect of other cultures and methods, which should be the modern culture default.

If nothing else, the West could at least roll back the educational reforms which erased their own heritage to regain those pieces. The West showing a bit of respect for itself certainly wouldn't be out of line either.

His head could have a lot of movement since he was in the air fast and is falling fast.

The power forward is falling head first.

He see's the ball which seems to be blurry maybe because he hurt his head and could be having blurry vision, go through the net perfectly like it was supposed to.

Could have moved his head in a way he wouldn't hurt it while falling

He may be trying to give a tip that you should always keep your head straight and look at the bright side of things.

the play is quick it is not like you can stop time like sonic, the flash, and quicksilver. So you have the follow the play in your head.

A player let the play of the game work out in his head. If the coach wants to call a pass, 3-pointer, or a defensive play. You can memorize multiple plays all at once.

i didn't think about this but perhaps this is why instead of the 3rd act being called chiron it's called black because he forces himself to become something like that bc of betrayal and not wanting to be hurt like that again

important quote but also the way that juan doesnt pass judgement, he tells him that you can be gay but dont let nobody call you no fag

I don't think he's thinking about the past here. I think he's thinking about reasons for why he can't do things or why he feels disconnected.

I might be a bad person but this last line made me laugh because it's such a radical left turn. The poem is talking up Corey on almost every line until "one calm summer night" he decides to end it. If you cut out the last two lines it would be a completely different poem.

can make this into a big list. "the head, around 30 to 38 pored scales covering its body, and its dorsal fins have 9 to 10 spines. I think you should also include a diagram with labels if you can find one--or make one yourself (not sure how you'd credit it correctly).

The data visualization examples in this article seem to primarily depict quantitative data. This makes me wonder how I can translate my qualitative data into categories that can be measured and depicted visually. For example, when I identify themes or common words and phrases that come up in my observations and notes, I could create a word cloud showing their frequency.

She cant see her legs in her memory of being in the fire. She doesn't know how to feel about this trauma she went through. When the speaker is reminiscing she is not sure if shes trapped in her head or if its a blessing she cant remember looking back.

Reviewer #2 (Public Review):

The manuscript by Diao et al. is an important extension of their eLife paper of 2017. Their development of new tools that allow them to follow Ca2+ transients in single muscle fibers over the whole animal through the behavioral sequence and also to independently monitor the Ca2+ transients in the endplates of the motor neurons that innervate these muscles. Their goal is to break down the movements that control the ecdysis sequence into elemental "syllables" and then to defined the role of these syllables in constructing progressively complex behavioral programs and as targets of neuropeptide modulation.

A crucial behavior that occurs during P1 in higher flies is the movement of the gas bubble but this event is largely ignored in the paper. Prior to pupal ecdysis, gas is expelled into the posterior puparial space and then actively translocated, via muscular contractions of the body wall, to the anterior end of the puparium during the latter portion of P1 (shown nicely in the author's 2017 Video). A detailed study by C.G. Chadfield & J.C.Sparrow (1985. Dev. Genetics 5: 103) of pupal ecdysis in Drosophila emphasized the importance of this translocation for head eversion. When they simply removed the operculum at the start of bubble movement, then the gas bubble could not push the animal backwards in the puparial case and head eversion could not occur. However, they saw normal pupation and head eversion if the removed operculum was immediately replaced and sealed down with petroleum jelly.

During translocation, the bubble moves in a fragmented fashion between the pupal cuticle and the puparium. Ignoring this movement leads to statements like on line 378 "Because pupal ecdysis is independent of environmental factors and executed in the absence of competing physiological needs, it is likely that its variability is intrinsic to the ecdysis network." For the pupating animal, its "environment" is the inside of the puparial case and the moving bubble is an unpredictable variable in this environment. The trajectory and route of bubble movement is not fixed, and it is likely that variation in sensory feed-back from the gas movement explains the motor variability and reduced stereotypy during P1. The role for proprioception during this phase is likely to inform the CNS of the progression of the bubble fragments. The author's finding that the blockage of proprioceptors suppresses the behavior progression could mean that this sensory information is needed to signal that an anterior space has been produced, and without this signal, the behavior does not progress to its next phase. This should be addressed in the text if not experimentally.

Another aspect of the background that is missing is considering earlier studies on the ontogeny of behaviors leading up to ecdysis/hatching. Notable are studies of the progressive construction of the flight motor program during metamorphosis in moths (Kammer & Rheuben 1976 J. Exp. Biol. 65:65.) and a similar feature of assembly of motor programs prior to hatching in Drosophila (Crisp et al., 2008 Development 135:3707). In the moth studies, complex motor programs were gradually assembled during ontogeny with motor neurons firing but without muscle contraction (as the authors see in prepupae during P0 - Fig 2C). A lack of excitation-contraction coupling in the moth prevents muscle movement through most of development. This suppression of contraction is essential because prior to production of adult cuticle, muscle contraction would rip the developing animal apart. The same requirement to suppress muscle contraction would be seen in fly prepupa until sufficient pupal cuticle has been secreted to prevent rupture from actual muscle contractions! This should be addressed in the text.

Besides not being explicit about how the syllables combine to build the eight basic movements, it is not clear how these basic movements then combine to support the major behaviors of each phase. This is seen in P1, where we see that swing and brace movements can co-occur (e.g., Fig 3D) but is a swing on one side always associated with a brace on the other? What are their phase relationships? Does their temporal association remain stable as the bouts progress? Another example is in Phase 3. There appear to be 5 basic behaviors associated with bouts in Phase 3. The example in Fig 1H shows double peak bouts in phase 3, and the bulk Ca data show a preponderance of double peaks. The different shapes suggest that there are different movements during the two peaks. Their discussion of P3 movements (around line 273), though, does not address this feature of the double peaks. The example in Fig 7A suggests that some movements, like the PostSwing occur at half the frequency of other movements such as the PostCon and AntComp. Is this the basis of the double peaks and how is that reflected in the movements that are finally produced? This should be addressed in the text.

One approach that I did not find useful was dividing the analysis into compartments - anterior versus posterior and dorsal-lateral-ventral. This may provide a way of generating some statistical analysis, but it did not illuminate anything about the behavior. The line between anterior and posterior segments seems to be arbitrary. Of course, it is important to know if there is directionality of movement [waves going anteriorly versus posteriorly], but beyond that, I am not sure what it adds. [Indeed, it made Fig 7 very confusing!] Also, I could not see a rationale for considering separate dorsal-lateral-ventral compartments. This should be addressed in the text.

not in the best head space

I remember hearing a story that Mozart wrote music "like a cow pees" (in one giant and immediate flood and then done) and this thought of large works of writing, etc. springing, as if fully formed from the head of Zeus, makes me wonder if there was a similar process Mozart used for music. How did he see it internally/mentally? Or had he simply played it so much or played with it to do this?

I've heard other writers mention similar things.

Brains On! (2021, May 3). What does 95% effective mean for a vaccine? We head to a stadium to learn! Warning: There are seagulls overhead! (Big thanks to @mariasundaram for help with this video!) Learn more about the #vaccines find family-friendly #coronavirus explainers at https://t.co/Zo9nORLEdI https://t.co/erPYnoKuZC [Tweet]. @Brains_On. https://twitter.com/Brains_On/status/1389293681669152769

THIS MEANS IF YOU F AROUND YOU GET YO HEAD BLOWN OFF, SO DON'T TRY TO FKING TEST ME

From the begining i feel like its a sort of survival place, the writer talks about streets and how its like- from the word streets i feel its talks about the difficulty of surviving the especially for those referred as negroes.

What a declaration!

as soon as she saw him, she has so many things running through head about him just by the first glanced.

This is a person who caught her eye, she found them. She is in the dominant role, that is probably why she is saying all this. I believe we are in her head, that she is not saying all this, but this is what matters.

When she saw him for the first time, thoughts immediately popped into her head about him.

This to me is saying she was looking at the person and all these thoughts came to her head about them being together.

Lichtenberg teasing around the idea of combinatorial creativity.

A fascinating quote in the history of the commonplace book on it's way to becoming the Memex.

I added more vivid details to this paragraph as before the details did not further the main idea of my essay. Before, I just described what I was seeing around me, without responding to it. I tried making the stimuli more meaningful as I linked the yellow caution tape to Covid-19 and how it changed my college experience, ironically as I write in a college library. The yellow tape stimulated ideas about what I was missing out on, in terms of the college experience, which I later address in the essay. By mentioning this, my final paragraph is more effective as it is linked to more paragraphs in the essay.

The expression "don't shoot the messenger" means to not be upset with the person that's relaying information, but to be upset with the person that said the information in the first place, so in this line, he's saying that people shouldn't be upset with him for saying what the voices in his head (which could be powered by his fears), people should be upset with the voices themselves.

The OCD he faces is bothering him, which must mean that he's aware that something is out of line or should be corrected.

He let the fame and acknowledgement get to his head, which caused him to acquire a bigger ego (which is one of the reasons why some people hate Eminem).

The person who is trying to save her from her fears and the voices in her head (her mental problems) thinks that she's crazy, maybe because she is refusing the help that she needs. She says the words "you think I'm crazy" twice to possibly emphasize that she is seen as deranged for being okay with these fears and mental struggles. .

Someone in her life believes that she is able to be rescued from her fears and the voices in her head, but she doesn't want them to waste their time and energy doing so.

She's learning to accept the voices in her head, whether the voices are spontaneous or are telling her bad things that she doesn't want to hear.

World Economic Forum: Global Trade Outlook (Live)

• Hypothesis.is Link https://hyp.is/W0tH_rL1EeuUcV8cMTIaww/www.youtube.com/watch?v=zHfgt4lpdt4
• 05/36
• They turn oif the "live chat" so I decided to go over the World Economic Forums Head.

Feel free to discuss with Hypothesis.is

https://youtu.be/wBPj0f5GEn4

Revisiting this some months later while thinking about programming in general, I realize that that what I want from a nextgen magic development environment are are live comments.

Take this _parseDefinition method, for example, and now compare it to the RSCAssembler's getRR method. The latter has a comment (with an interesting diagram), and the former does not. Suppose even that there were a comment for the _parseDefinition method—it might contain a snippet that's meant to be sample input to help visualize what the method is doing—there would still be the matter of needing to simulate the program in your head. Really, what I want is to be able to write out a snippet and then while reading the code here, anyone can mouse over each statement in the method definition and see what effect it has—say a caret moves along, pointing at the scanner "tip", and visually chunking the tokens.

One should also be able to dynamically modify that snippet—let's say they wanted to "link" the live, scrubbable execution to a particular point in Fig. 1 (the fixT entry instead of fixP, for example)—in this case that snippet should "take over" the live comment, so we can concretely visualize execution of it instead. We should go further than that, even, and not even require the reader know which part of Fig. 1 is actually handled by this section of the code. We should be able to dynamically discover, based on the Ctrl+Space activation, that _parseDefinition is used to handle the fixP, fixD, and fixT lines, and then then reader can point to them (like the difference between a command-line interface where you have to know what you're allowed to do and how to say it, compared to a graphical interface that visually communicates the program's affordances).

The closest I've seen to this are the Moonchild editor :https://harc.github.io/moonchild/ and REPLugger :https://github.com/Glench/REPLugger.

She could be replaying the way the relationship goes in her head over and over.

Is the pain he has experienced.

that he probably saw a girl calling for help

This explains the pains of the poet, he also explains where his tears were coming from.he also explains what he picture's the girl to be like this could be related to seeing one's loved one suffer

he was a young boy, he had no perception of race but the adults around upheld the standards of race. they treated him better because he was part white, the narrator realized this but was confused as a kid to why he was getting treated better and why he was called white.

A good introduction to pull in the reader.

this emphasizes on the way we were raised as kids. Even though we could have had different perceptions than our elders or adults, we didn't question it because in our head, they were the mature ones.

she is offended that he picks her up with no head lights this shows that he doesn't care that much about her because he doesn't bother to turn on the headlights.

this puts a image in the readers head of something nasty, hoping we wont forget about our dreams?

Reviewer #2 (Public Review):

In this manuscript, the authors use in vivo calcium imaging of many individual neurons to investigate how dopamine regulates striatal dynamics. They aim to determine whether manipulations of dopamine signaling, on acute and chronic timescales, change the rate of activity in individual neurons, but also how the number of neurons activated (the size of the ensemble) may also change. This is a very important question, which is challenging to address with traditional methods in mouse models. Single-unit electrophysiology, especially in mice, yields modest numbers of neurons (typically <<50) during any one recording. In addition, acute electrophysiology tends to be biased: higher firing rates make it much easier to detect single units. In relatively quiet brain regions, including the striatum, these factors make it very hard to compare the total number of neurons recruited during a specific behavior across conditions.

One of the major strengths of this manuscript is that the authors make use of a strength of their method (the ability to capture activity across hundreds of neurons), rather than trying to use it merely as a surrogate for a traditional method (by measuring the rate of activity). Another strength is the alignment of neural activity to specific behavior (locomotion), and attempts to control for changes in overall behavior with each of their dopamine signaling manipulations.

Weaknesses, which the authors to some degree acknowledge, include the fact that calcium imaging is not equivalent to action potential firing; changes in the activation across a population may represent a change in the firing rate or pattern. For example, a doubling of the number of neurons that are "active" during a behavior may represent a shift of completely silent neurons to firing above a certain threshold rate, and/or a shift to burst firing mode, without a change in overall rate. Another weakness, partly driven by the head-fixed/treadmill configuration, is the laser focus on locomotion (starts, stops, velocity), though the dorsolateral striatum is likely to regulate other behaviors (grooming, licking, rearing, etc). Finally, again related to their methods, the findings are observational, shedding minimal light on the mechanisms (direct effects on SPN cell bodies? Indirect effects via local GABAergic signaling, dopamine terminals, or glutamatergic inputs?) by which dopamine signaling manipulations lead to changes in SPN ensemble activity.

Despite these weaknesses, I suspect this manuscript, together with other recent studies, will change how other basal ganglia physiologists think about neural activity. Much as the field has emphasized dynamics and synchrony as potential ways neural activity regulates behavior, hard data regarding the spatiotemporal activation of neurons is relatively new. It is also likely to be thought-provoking for investigators working on Parkinson's Disease, as it suggests more cellular/mechanistic lines of research are needed to explain the massive changes in dSPN ensemble size seen in healthy vs 6-OHDA-treated and L-DOPA treated mice.

Reviewer #1 (Public Review):

This study investigates roles of DA modulation in projection neuron ensembles in DA-intact mice and Parkinson's disease mouse model using two-photon calcium imaging of direct and indirect SPNs (dSPNs and iSPNs) simultaneously in head-fixed mice locomoting on a freely rotating or motorized circular treadmill. The study begins with careful validation efforts related to their particular imaging conditions and reporter usage. Major findings are: 1) In DA-intact mice, they found that reducing DA receptor signaling by administration of D1/2R antagonists increased iSPN ensemble size (fraction of imaged iSPN active during locomotion) and decreased dSPNs ensemble size, resulting in an imbalance of striatal outputs in favor of indirect pathway. Consistently, elevating DA receptor signaling by D1/2R agonists yielded a dose-dependent imbalance in favor of direct pathway. Interestingly, at one intermediate dose of D1/2R gonists, iSPN ensemble size remained unchanged while dSPN ensemble size increased, whereas at higher doses, both iSPN and dSPN ensemble sizes shrunk. They also showed that reward-induced and nomifensine-induced DA increase recapitulated the low and high dose effects of DA on SPNs, respectively. 2) In dopamine-depleted Parkinson's disease mouse model, the authors found that 6-hydroxydopamine (6-OHDA) treatment reduced dSPNs ensemble size acutely (within 24h) and chronically (after 30 days) and increased iSPNs ensemble size acutely. However, the active iSPN ensembles returned to pre-lesion levels within one week. Overall, ablation of SNc DA neurons biased the striatum output toward indirect pathway. Lastly, they evaluated the influence of L-DOPA on SPN ensembles in DA-depleted mice and found that l-DOPA increased the dSPNs ensembles by 10 fold and reduced the iSPN ensembles to below pre-ablation levels, resulting in strong bias toward direct pathway, a finding they suggest may relate to levodopa-induced dyskinesias. Together, this study introduces data to support the concept that SPN "ensemble size" may be relevant for long-standing pathway balance ideas concerning striatal circuitry in the control of normal movement and its demise in PD.

Author Response:

Reviewer #2:

The manuscript "Adult Stem Cell-derived Complete Lung Organoid Models Emulate Lung Disease in COVID-19" by Das and colleagues introduces a new model system of airway epithelium derived from adult lung organoids (ALO) to be utilised for the study of COVID-19-related processes. In this manuscript two main novelties are claimed: the development of a new model system which represents both proximal as distal airway epithelium and a computationally acquired gene signature that identifies SARS-CoV-2-infected individuals. While interesting data are presented, the novelty claim is questionable and the data is not always convincing.

Strengths:

Multiple model systems have been developed for COVID-19. The lack of a complete ex vivo system is still hampering quick development of efficient therapies. The authors in this manuscript describe a new model system which allows for both proximal and distal airway infectious studies. While their claim is not completely novel, the method used can be used in other studies for the discovery of potential new therapies against COVID-19. Moreover, their computational analyses shows the promise of bioinformatics in discovering important features in COVID-19 diseased patients which might elucidate new therapeutic targets.

Weaknesses:

Although the paper does have strengths in principle, the weaknesses of the paper are that these strengths are not directly demonstrated and their model system is not completely novel. That is, insufficient analyses are performed to fully support the key claims in the manuscript by the data presented. In particular:

The characterisation of the adult lung organoids and their monolayers is insufficient and sometimes incorrect. Their claims are based on contradicting data which includes cell composition in the culture system. Therefore, the claim of a novel model system seems invalid and rushed. Moreover, the characterisation of a new gene signature is based on this model system which has been infected with SARS-CoV-2. The infection however is hard to interpret and therefore claims are hard to validate.

First of all, we thank the reviewer for a very thoughtful and in-depth review that inspired us to do additional analyses to address the criticisms that we believe are not just fair and justified, but also constructive. Coming from a thought leader in the field, they also squarely point at essential areas where we needed to make improvements with additional analyses. For that, we are grateful.

There appears to be three major critiques:

(i) NOVELTY: The reviewer questions whether the model itself is novel, and asked how this is any different from the previously published manuscript (Lamers et al., in EMBO J (2021)40:e105912) describing lung organoids with mixed cellularity, also claimed as complete with proximal and distal components that was publicly released after ours was submitted to eLife.

(ii) INCOMPLETE: The reviewer noted that the model system was not fully characterized to have reached that potential and the impact of culture systems on cell composition and such details were incompletely analyzed (hence, rushed and incomplete).

(iii) CLARITY ON GENE SIGNATURE: Characterization of organoids with new gene signature was added to interpret.

Overview of how we tackled these three points head on:

(i) NOVELTY: As for what is novel in our model (i.e., ALO), and how does it compare to the model described by Lamers et al., in EMBO J, 2021, we provide metrics for which is closer to the human disease when infected with SARS-CoV-2. Figure 6H was added.

(ii) INCOMPLETE: We agree about the ‘rushed’ aspect. We were working amidst a pandemic to race to the finish line. But during the revision we could add much more characterization data, which we hope mitigates the concerns raised by this reviewer. Three new figures (Figure 2- Figure Supplement 3-4-5) and some IF panels in main Figure 3 were added.

(iii) GENE SIGNATURE: As for the characterization of the SARS-CoV-2 infected ALO-derived monolayers using a new gene signature, we apologize that we might not have written with sufficient clarity as to what was the source of the signature. In the revised version of the manuscript, we have now explicitly stated in the edited Figure panel 6A that the signature (166-gene viral pandemic signature, a.k.a. ViP) was derived from human clinical samples, after a comprehensive analysis of > 45,000 datasets. This paper has been accepted in eBioMedicine in April 2021, and the preprint is available in BioRxiv 2020-PMID: 32995790.

Reviewer #3:

The authors have developed a new culture method to expand adult lung cells in vitro as 3-D organoids. This culture system is different from previous organoid cultures which include either bronchiolar, or alveolar, lineages. Rather, the authors attempted to preserve both lineages over long-term passaging. The 3-D cultured organoids can be dissociated and re-plated as 2D monolayers, which can be either cultured immersed in medium or in air-liquid interface (ALI) conditions, exhibiting a different bias towards alveolar and airway lung cell types respectively. The 2D monolayer cultures can be infected by COVID-19 virus and showed a progressive increase in virus load, which was distinct from iPSC- derived alveolar type 2 (AT2) cell and bronchiolar epithelial cell culture control infections. Through bioinformatics analysis, the authors were able to show that their monolayer cultures acquired similar immune response features to an in vivo COVID infection dataset, indicating that this culture system may be suitable for modeling COVID infection in vitro. It is particularly interesting that the bioinformatics analyses suggested that this adult human lung organoid system, with both airway and alveolar phenotypes, showed greater resemblance to the transcriptional immune response of severely COVID-infected lungs than either cultured cell type alone. This aspect of the manuscript strongly suggests that the authors' approach of developing a mixed lung organoid model is an extremely good one.

However, the data presented in figures 2 and 3 cast serious doubts over the long-term reproducibility of the organoid system. That individual organoids contain both airway and alveolar lineages has not yet been convincingly demonstrated (Fig 2). In addition, bulk RNAseq experiments illustrate that the overall cell composition of the cultures drifts significantly during long-term passaging (Fig 3). Due to this variability, the organoids' ability to act as a suitable model for viral infections that would be amenable to drug screening approaches is also questionable.

We thank the reviewer for the generally positive nature of the comments. The reviewer made some key and thoughtful suggestions on how to improve the manuscript; we greatly appreciate the effort and time that went into making them. Besdes the encouraging comments and the suggestions, the reviewer also raised some criticisms that are along the same lines as those that were raised also by Reviewers 1 and 2. We have tried our best to address these criticisms and agree that mitigating these are essential for widespread acceptance of the model by others.

Another foreshadowing, but with Zenobia coming out on top instead. The first part of this reminds me of Priscilla first making an appearance and being shy. Then, she started to blossom into someone that everyone loved. Everyone lit up when she came around. There was no denying that she had contributed to the happiness in Blithedale. However, Zenobia sort of threw this in her face when she told this story with the veil being thrown over the girl's head. It's also interesting that she then places the cloth on Priscilla's head, as if to replicate the story.

There is a Hispanic story which is very similar to this one. A man, named Carlos, meets a woman walking by a cemetery. He instantly has an infatuation, and wants to go on a date with her. She tells him to meet her at the same time the following night and she will think about it. Carlos returns the next evening. They spend a lot of time together that evening, and she tells Carlos she will send him a letter with a new place to meet the following night. The next day, Carlos finds the letter in his mailbox (he never gave her his address but he doesn't think of this) and shows his cousin the letter. The cousin is in shock, because the signature reads, Rosa Gonzale, who is a woman who died in a car accident the year prior. Carlos ignores his cousin's warnings, and goes to meet Rosa.

"That night as Carlos hurried to the cemetery Diego followed, certain that his cousin was in over his head. Carlos bounded over to Rosa. “At last, we go out!” he cried to her. “But first, my love, show me your face!”

At his words, Rosa pulled aside the veil. Back at the gate, Diego gave a gasp of shock, for she had the desiccated face of a skeleton. He was frozen to the spot by the power of the evil specter, unable to warn Carlos. Looking down, Carlos only saw the glamour the ghost was projecting. As the skeleton’s withered arms trapped him, the veil on his eyes was lifted and he realized in one heart-stopping moment the abomination he was kissing. The ground opened up, and with a laugh of triumph, the specter pulled him down and down into her tomb. The earth closed over Carlos and Rosa.

Diego, freed from the ghost’s spell, ran into the cemetery, shouting his cousin’s name in terror. But it was too late. Carlos was dead—locked for all time in Rosa’s arms."

Rosa took off her veil and instantly Carlos was under her spell and taken into the grave with her. While Zenobia's story does not quite follow the exactly storyline, it is very similar. The Veiled Lady cursed Theodore with the inability to be happy for the remainder of his life. He could have had a beautiful bride, but did not trust her. Carlos was eager to meet his bride, but it resulted in his death.

31:40 — Analog collections are managed at the collection or box or folder level. Digitizing an item in a folder almost mandates item-level metadata.

55:49 — Turn the formal digitization model on its head...put as much out there as possible in whatever low quality you can do, and then see what rises to the top.

The author's use of diction gives the readers a better understanding about who the people in the war actually were.

I think this is applicable to any sort of learning. By breaking it down into something that we can easily condense you

1. Maintain the motivation to keep working through it and it's short enough that you're able to quickly get the rush of having completed something.
2. By condensing some concepts into an overarching concept (or maybe still concepts) you don't have to hold so many things in your head when using the concept elsewhere.

Not sure about the slip into the conditional if it's a recommendation. In my head I have it as: "It is recommended that: 1. The Network [should] take... 7. The Network facilitate..." The "could" I find awkward and a good deal weaker.

Nas is thinking back to about a violent insurrection of sorts to gang related

Could be referring to gang activity and he started a war before running off

Basically saying that hes about to start a war in the streets by killing somebody

Trying to start a movement to fix these systemic issues

Represents gang violence, that he is exposed to.

the term catch a body has many different meanings. For this example its about killing someone

Scarface let the fame and things that came with the fame get to his head, so is Nas saying that the pain gets to his head?

scarface was this young man who became something crazy and the money got to his head the power got to his head the girls go to his head and Scarface ended up dying

This goes along with the last line. She is putting a picture in your head of her standing staring at a sunset, with a nice dress on. She doesn't want the relationship to be viewed as "oh, why did we do that" but instead "yeah we did that, and we have some good memories out of it."

There's constant conflict.

Following with this line after declaring it a useless task makes it seem like she was taught to give up on her independence as a woman. To keep sewing makes her inferior to the woman she wants to be..

Who exactly is after Mohsen and the uncle?

This part where the mother is murdered in the bathroom is scary, because it shows that no one is safe

or to make the head dancer man by her amazing dancing

emotionally distress

It looks like they are well known big time in the area.

I really like this differentiation between what autofiction is versus autobiographical fiction. What may see like a small difference actually makes a difference in the way it is assessed by critics, as stated by the author. Other than the reasons stated, I'm curious about what these kinds of differentiations mean for authors when they are decided what to write. I feel that it might be kind of difficult in your head to imagine if this idea you are having is "new" or "traditional."

It sounds like a fish. They are lifted out of the water, gripped hard so not to slip away, its head is chopped off, and it is eaten once the scales are removed.

In a relationship, a woman looks forward to being treated like a queen. The man ,being the king, would have to be a leader of the family and head of the house.

This makes me really mad and sad. There's tears my eyes. I don't know why. It's really hard to read and not feel something there.

why was the woman the punching bag? Why did they not eat what they sold to the whites?

Someone else is steering your life. What this snippet says is that there is no you. Just experience

This went over my head, as other professions have code of ethics but never wondered in the computer science world what or if there were a code of ethics. As this is important to the field. Taking a look at other professions such as teaching where there is no code of ethics, teachers are punished for trying to help students outside of school. By having a code of ethics we might be able to look at situations and figure how to approach situations without having to go through a self moral dilemma.

The first sentence seems to makes an assumption that, that's just the way it is - to translate.

Power saving from downgrading QPI bandwidth can provide extra head room for core scaling, but increase data access latency by cpus

“It's a delicate balance between showing right away that you can make a difference, and also being a sponge and soaking up as much information as possible and that you're hungry to learn from everyone. When you're juggling this tradeoff in your head, I'd err on the side of showing in the first week or two that you can make a tangible impact because startups thrive on speed,” says David Mok, Director of Content and Partnerships for Labelbox.

“If you work with your manager on strong goals, this should be baked into your onboarding — but startups also usually don't have a lot of structure yet. Actively seek out a few pockets where you can make an impact. Hint: it's usually found in the places of the business that are not fun or sexy. Examples could be spending some extra time to QA a feature, writing multiple versions of marketing copy to see what looks best, or creating a sensitivity analysis or quick spreadsheet model for a business decision.”

Carrying bucket of water on her head

I'm passionate about the cause. As a British-Brazilian person, I am seeing the dismantling of public services on both sides of the Atlantic Ocean. In 2015, before the coup in Brazil, I was working for The Guardian and spoke to the head of communications at the Brazilian Ministry of Health. The NHS, he said, was and had been the blueprint for Brazil's public health system. Even though the country has just a fraction of the resources - I've lived first hand what it means for everyone to have access to free healthcare and higher education.

When lessons are learned the hard way, it’s a good time for introspection and team-wide reflection to fully capture what went wrong. “I talk a lot about this actually in my book — obviously a lot engineering-minded folks are accustomed to this notion of the ‘five whys’ of blameless post-mortems. If the site has an outage, you don't blame the person who wrote the bug and deployed it to prod. Instead you ask the question, ‘How do we let a single mistake by a single human being actually take down our website?’” he says. “And instead of blaming that person for being a human being, you blame the system and you get to the true root cause. The root cause may be the lack of testing. You might ask, ‘Why don't we have testing?’ Well, we don't educate developers on how to write good testing. Or maybe we don't have a good investment in infrastructure to do testing. And when you get to the true root cause, then you can make systemic change,” he says. “When we had that Uber situation, for example, we did a blameless post-mortem and got to the true root cause.” Lawson’s seen plenty of companies leave post-mortems behind when every piece falls into place — but he’s a big believer in flexing these same muscles, even when things go well. “Usually post-mortems is the word you use to describe analyzing the things that don't go well, but we do post-mortems when things go well, too,” he says. “That is the way in which you continually build this muscle of analyzing the outcome and asking what all of the inputs were that led you there and try to do your best in the moment when everything's fresh in your head to learn and to capture knowledge,” he says.

I feel like the author is basically arguing that Atheism victory was no victory and just a false victory. Secularity became a assumption and not some intellectual victory. Religion thrown on the side.

Obviously this is true to some extent however, religion has been fighting a war with science for hundreds of years and has clearly intellectually lost. The vast majority of religions do make empirical scientific claims

1. dualism of some kind
2. life after death
3. claims of the age of the earth
4. man being made at once.
5. claims on how the earth or universe was founded
6. archeological and anthropological claims
7. claim of demons and exorcisms

(1) there is no central model that is the contrary to physicalism. There is no dualistic science or soul science. There is however psychology, neuroscience and cognitive science. (2) if physicalism is true that minds depend on bodies and when bodies die the mind dies then their is no life after death (3) geology finds the earth is much older then most religions claim (4) biology and paleo-anthropology finds a gradual evolution of man, not intelligent design (5) cosmology and astronomy does not find the belief common in most religions that the earth is the center of the universe (it's a tiny blip) or that it's flat (Vedas argues this) (6) we haven't found a great solomonic kindgom or other major claims of religions (7) exorcisms have little evidence (can be explained as communal reinforcement & self-deception) nor demons as the source of diseases.

I have head the claim before (I think some post-modern philosopher wrote about it) that Atheism came about due to theologians throwing God further and further back. Descarte & Locke both christian fundamentalist put our body of knowledge on a firm rational and empirical basis. Kant had to throw God into the noumenon, from their Atheism came easily with the likes of Feurbach arguing it is projection of human ideals

Im not sure I believe both stories but this is kinda a inverse of that.

Perhaps the mother is trying to find a talent she has

Some of these tests seemed unnecessary, but I get that the mother was just trying to expose any hidden talents.

• India has made a fresh proposal at the World Trade Organisation (WTO) for the establishing the mechanism of true vaccine internationalism.
• (WTO) is under pressure to consider the India’s proposal of Trade-Related Aspects of Intellectual Property Rights (TRIPS) waiver on Covid-19 vaccines.
• General Agreement On Tariffs And Trade (GATT)
• Nigerian economist Ngozi Okonjo Iweala has been appointed as the head the World Trade Organization.
• India and 17 countries face U.S. Anti-Dumping Tax

in order for us as a society to move forward and progress, it first comes with stopping the usage of a word that in it's infancy had such a negative connotation, idk why we still use it today.

I am glad he apologized

a great explanation of how to role play a character

A possible central idea could be it takes many practice and hard work before you become successful because se had flying lessons, and to be able to have lessons, she had to work at a telephone company called Los Angeles Telephone Company.

I do think if someone is nodding their head in agreement and they really don't understand what is being said, it might be their fault for not speaking up and making it known they dont understand what was said.

I agree, why would someone risk their life just for sport?

The author hadn't acknowledge the word until it was used in a way that disrespected her or used in a negative manner, which to me shows how far in between it is to see the word being used in a negative manner now, becasue usually people use it in a positive connotation. Such as today's rappers.

oh wow, this is a prime example of folk evolutionary reasoning

I always wondered how a meme becomes famous and how it just works in a way that other people can play off of it and recreate it over and over to expose and market the purpose even more. Is it a by chance natural type of thing. Or whoever created it knew that it was going to hit the nail right on the head.

Starting off by stating that sewing is a useless task, combined with the weighed down dreams, seems like negative reinforcement set upon the speaker by others, with the words "it seems," it can be suggested that the uselessness of the task isn't her own opinion.

Here, she's talking about dreams that she cannot achieve because of her race and gender. But I like this line because it does a good job at portraying the mind while doing menial tasks over and over again. Without going into too much detail, I've recently had a job that was very repetitive with little to no social interaction. While it was fine at first, I was soon left alone with my thoughts while I did my tasks without thinking. Soon enough, bad, personal thoughts entered my brain and it was impossible to escape them. I couldn't listen to music or podcasts to stimulate my brain so I had to endure it. It wasn't a good feeling and I think many people can relate to being trapped in your head while doing the the same boring tasks over and over.

This being the first part of the line following "my head weighed down with dreams" makes me wonder if this is a dream to the speaker, if this is that weight, in the sense of an ambition.

During the time frame, after enslavement ended, a lot of Black women could only get jobs as seamstresses whether that's what they wanted to do or not.

Tori slowly realizes that his mother stole a wallet from that customer and tried to buy the opium with it.

I think this shows how strong Malala is, and despite having such a risk she's still risking it, so she can stand up for her beliefs. (which many girls may not do due to them having a whole life ahead)

This shows that if Malala can get shot in the head and she recovered then, she can do anything.

she thinks he wants to marry her. But this could be harmless flirting? she worries about intention

This expository text uses many different vocabulary strategies. I loved how the vocabulary is bolded in each section. This signals unfamiliar words to students. One strategy that students could use to further their comprehension of their new vocabulary words is through technology. After reading the text, students could look at videos that explain the meaning of each word in ways that they can see. Real world learning! Chlorophyll would be a good word to research further because it is a hard concept to visualize in your head. It would make more of an impact on students if they were able to see it.

i think its super important to volunteer for something that you actually care about and want to help out with, otherwise if you don't like what you are volunteering about, then you wont put a lot of love into it

Why did America do this to all these people? This is hurts a lot and the people suffered a lot so I think America shouldn't have done what it did.

How did survivors live like this with all their skin peeling off? Did many die after this event or were their skin healed? Either way this was a horrible time for the Japanese.

Central idea: When you punish a country, don't punish it's innocent people.

That's painful, why did America do this?

CENTERAL IDEAS: War is brutal. Which ever side had started this war has certainly been holding a big grudge against the other side, to be killing innocent people.

When I was completing the survey, I had a hard time wrapping my head around the concept of systems with video games, game design and gaming literacy. I liked this whole paragraph on systems and it helped to shed light on a concept I was struggling with.

The head of the family gets their own table and eats alone which shows respect but I would feel lonely if I was eating alone I would rather enjoy my food with company.

This demonstrates that the husband is superior so he gets to eat by himself. I wonder if they liked being alone while eating. I would not want to eat alone even if I was superior. It is similar to the manual "Of Domesticall Duties" where it mentions that the husband is the head of the family and is superior while the wife is the heart and is still considered high, but not as high as the husband. It follows the same lines here as the husband gets to eat alone because of his superiority and the wife has to eat with the slaves as they are considered inferior.

really helps you create an image in your head, very detailed

This is restating the first lines and Hughes did this to emphasize the point being made here. Langston Hughes was in agreement with Washington's philosophy that the work must be put in to attain the dream they so longed for.

He is restating previous lines of the next to bring notice to them and show how important they are.

By emphasizing this line at the begging i end shows us that Hughes wants us to recognize the fact that Washington views aren't all that bad.

We get to see how Hughes agrees with Booker in the sense that by accepting your fate will not only help you as a person, but overall lead to the black community success in the long run.

The repetition of this line in the beginning and now the end means the author wants you to pay attention to it. Putting it here after the last quote means a lot. It shows that Washington deep down has the same goals as DuBois for example. He wants the perfect picture too, he just wants to make sure they are doing it the right way.

He believes that is strategy will not only help the individual, he believes it will help the entire black community by proving their worth.

Detailed characteristics, these were helpful to providing an image of what Oscar may look like and how he acts

I still can’t wrap my head around the fact that they would do genital manipulation on young girls and force them to marry a stranger man that is old enough to be their father. I never knew there were places that still followed this “standard” norm, It was very heartbreaking to hear. All these young girls have so much potential and providing education will allow these womens to live a better life and not depend on anyone.

SciScore for 10.1101/2021.04.13.21255427: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

No key resources detected.

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Our study has several limitations. First, safety concerns and the lack of a BSL-3 level resources precluded the use of viral cultures to determine viability of virus, but nucleic acid of target pathogens can be consequential [19]. Furthermore, lack of culture-based assessments is not unique to our study [4-9]. Second, sponge sticks may be better at capturing pathogens from larger environmental surfaces such as floors and tray tables. These collection devices were unavailable and could not be used in automated PCR reaction tubes. Third, the commercial remediation service and EVS were not compared under identical conditions, but our main objective was not a direct head-to-head comparison of EVS vs contracted remediation service. Despite these limitations, this report is relevant to nursing homes and hospitals as the findings can assist in allocation of scarce cleaning and disinfection resources for maximum impact. Furthermore, studies of the survivability of the virus on fomites have been criticized as lacking real-life generalizability [22, 23]. Data in this report were obtained from a ‘typical’ general hospital and nursing home, representative of the type of settings (i.e. general/community/non-university-based hospitals) where much of health care is delivered. Studies for these settings may be underrepresented in research because most are carried out in large university-based hospitals. In summary, surface contamination with SARS-CoV-2 RNA significantly differed based on typ...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

without evaluating the viability of these marketing ideas, it's important to see that we were grounded in the commercial world.

"head vs. heart", implying reason over emotion. Think of how scary monsters in movies become less scary when we have a name for them. Without a name, we can only rely on the "heart" sensory impression (terror, uncanniness), but if we have a name for it, we can put it in a category.

example of the uncanny.

Reminds me of Maria Anderson's new ESIL learning objective scale.

Not only does the writer talk about writing as a form of expression, but the writer excessively describes the wallpaper in her room, how they crawl, and jump off the paper. Lots of personification. What is the importance of the personification? Does this show her creativity in her head, and he has no way to express her ideas, which is making her unwell? Is there a deeper meaning to this personification and relation to "paper"?

Interesting

This paragraph does a good job of emphasizing the unfortunate situation he is in, painting a descriptive picture on what he has to do as a "horrible vermin" to get out of bed

Continues to struggle with the idea of getting out of bed to go work, but rationalizes it finally in his head and has a plan

Does this mean that we are now capable of surviving in space in a long period of time?

Reviewer #2 (Public Review):

In this study, Huang and colleagues recorded local field potentials from the lateral habenula in patients with psychiatric disorders who recently underwent surgery for deep brain stimulation (DBS). The authors combined these invasive measurements with non-invasive whole-head MEG recordings to study functional connectivity between the habenula and cortical areas. Since the lateral habenula is believed to be involved in the processing of emotions, and negative emotions in particular, the authors investigated whether brain activity in this region is related to emotional valence. They presented pictures inducing negative and positive emotions to the patients and found that theta and alpha activity in the habenula and frontal cortex increases when patients experience negative emotions. Functional connectivity between the habenula and the cortex was likewise increased in this band. The authors conclude that theta/alpha oscillations in the habenula-cortex network are involved in the processing of negative emotions in humans.

Because DBS of the habenula is a new treatment tested in this cohort in the framework of a clinical trial, these are the first data of its kind. Accordingly, they are of high interest to the field. Although the study mostly confirms findings from animal studies rather than bringing up completely new aspects of emotion processing, it certainly closes a knowledge gap.

In terms of community impact, I see the strengths of this paper in basic science rather than the clinical field. The authors demonstrate the involvement of theta oscillations in the habenula-prefrontal cortex network in emotion processing in the human brain. The potential of theta oscillations to serve as a marker in closed-loop DBS, as put forward by the authors, appears less relevant to me at this stage, given that the clinical effects and side-effects of habenula DBS are not known yet.

Detailed comments:

The group-average MEG power spectrum (Fig. 4B) suggests that negative emotions lead to a sustained theta power increase and a similar effect, though possibly masked by a visual ERP, can be seen in the habenula (Fig. 3C). Yet the statistics identify brief elevations of habenula theta power at around 3s (which is very late), a brief elevation of prefrontal power a time 0 or even before (Fig. 4C) and a brief elevation of Habenula-MEG theta coherence around 1 s. It seems possible that this lack of consistency arises from a low signal-to-noise ratio. The data contain only 27 trails per condition on average and are contaminated by artifacts caused by the extension wires.

I doubt that the correlation between habenula power and habenula-MEG coherence (Fig. 6C) is informative of emotion processing. First, power and coherence in close-by time windows are likely to to be correlated irrespective of the task/stimuli. Second, if meaningful, one would expect the strongest correlation for the negative condition, as this is the only condition with an increase of theta coherence and a subsequent increase of theta power in the habenula. This, however, does not appear to be the case.

The authors included the factors valence and arousal in their linear model and found that only valence correlated with electrophysiological effects. I suspect that arousal and valence scores are highly correlated. When fed with informative yet highly correlated variables, the significance of individual input variables becomes difficult to assess in many statistical models. Hence, I am not convinced that valence matters but arousal not.

Page 8: "The time-varying coherence was calculated for each trial". This is confusing because coherence quantifies the stability of a phase difference over time, i.e. it is a temporal average, not defined for individual trials. It has also been used to describe the phase difference stability over trials rather than time, and I assume this is the method applied here. Typically, the greatest coherence values coincide with event-related power increases, which is why I am surprised to see maximum coherence at 1s rather than immediately post-stimulus.

Kiowa uses the Bible as a source of protection, this is why he sleeps with it.

Kiowa sleeps with the Bible beneath his pillow hoping it could protect him from all evil and danger.

intangible

The thought of knowing that you might die i would be terrified just like them.

Kiowa feels like in terms of already losing someone important that now he must seek protection from the New Testament.

Kiowa feels that the bible is important to him; it represents hope in the situation, something to look up to.

The author explains tot the reader what the people the main character is around is like.

This is a very in depth take on the people in this squad, we see how each of them have their own necessities of items in which they take into war. This shows that each person has a depth to them, even Ted Lavender who was apparently deceased due to gunfire.

Lt Cross' is head over heels for Martha and he wishes for her to feel the same. I don't think Martha knows that he likes her since they are communicating through letters to me it seems one sided because she is trying to make sure that he is safe and I don't think he writes back

What was Hitler thinking when he killed the Jews. Why did he hate them?

the name of the game is to express the concepts in our head

Author Response:

Reviewer #1:

The authors demonstrate in this study that it is possible to train mice to perform a challenging tactile discrimination task, in a highly controlled manner, in a fully automated setup in which the animals learn to head-fix voluntarily. A number of well described tricks are used to prolong the self-fixation time and thereby obtain enough training time to reach good performance when the decision perceptual decision is difficult. In addition the study establish that this experimental design allows targeted silencing of relatively deep brain areas through a clear skull preparation.

It has already been demonstrated that mice can perform voluntary head-fixation and can do behavioral tasks in this context. However, this is the first time this methodology is applied to first to a tactile task and second to a task that mice learn is thousands of trials. Another advantage of the present technique is that it is fully automated and allows training without virtually any human intervention.

The demonstration that optogenetic silencing can be performed in this context is nice but not very surprising as already done in other contexts. Nevertheless it is an interesting application of self head-fixation. The authors should make sure that a maximum of information is available relative to the efficiency of the silencing (fraction of cells silenced) and about its impact on the behavior (does it result or not in a complete impairment?).

We have improved presentation in various places of the paper to provide more information about the optogenetic manipulation. We added new analysis of the fraction of neurons affected by photostimulation (Figure 8E). We also analyzed the impact on behavioral performance relative to chance performance (Figure S4A and S6). We compared the effect size to prior studies (Figure S4) and we discuss the interpretation of effect size (Discussion, page 22).

In the power range tested in this study, photostimulation did not reduce performance to chance level (Figure S6). One limitation of the optogenetic workflow is the interpretation of behavioral deficit effect size. We examined this issue in ALM, a brain region from which we have the most extensive data. In previous studies, we have shown that bilateral photoinhibition of ALM results in chance level performance (Li et al 2016, Fig 2b; Gao et al, 2018, Extended Data Fig 6b). Here, mice performance was above chance during photoinhibition of ALM (Figure S4). This difference in effect size likely resulted from incomplete silencing of ALM. The photostimulus intensity used here was much less than those used in previous studies (0.3 vs. 11.9 mW/mm2). In addition, a single virus injection was not sufficient to cover the entire ALM. Thus a partial behavioral effect could be due to incomplete silencing of a brain region, or partial involvement of the brain region in the task. Given this limitation, we caution that the function of a brain region could only be fully deduced in more detailed analysis and together with neurophysiology. The workflow presented here can be used as a discovery platform to quickly identify regions of interest for more detailed neurophysiology analysis. We now better highlight these points in the Discussion.

Reviewer #2:

Hao and colleagues developed an automatic system for high-throughput behavioral and optogenetic experiments for mice in home cage settings. The system includes a voluntary head-fixation apparatus and integrated fiber-free optogenetic capabilities. The authors describe in detail the design of the system and the stages for successful automatic training. They perform proof-of-concept experiments to validate their system. The experiments are technically solid and I am convinced that their system will be of interest to some laboratories that perform similar experiments. Despite the large variety of similar automated systems out there, this one may prove to become a popular design.

The weak side of the work is that it is not particularly novel scientifically. The system is complex but there it is not an innovative technology. The body of the study has too many technical details as if it is a Methodological section of a regular manuscript. There are bits of interesting information scattered around the paper (like the insights about the strategy mice use, which stem from the regression analysis), but these are not developed into any coherent direction that answers outstanding questions. The potential advantages of this system compared to other systems is marginal. In my eyes, the fact that manual training is so similar to the automatic one is not only a positive point. Rather, it signifies that the differences are mainly quantitative (e.g. # of mice a lab can train per day, etc). Thus, even as a methods paper, the lack of qualitative difference between this and other methods weakens it as a potential substrate for novel findings.

The automated workflow presented here significantly boosts the yield and duration of training to rival and slightly surpass that of manual training for the first time (new Supplemental Table 1). We think this degree of automation is an important technical advance. We show that the workflow can significantly scale up the throughput of optogenetic experiments probing behaviors that require thousands of trials to learn. This enables efficient and systematic mapping of large subcortical structures that are previously difficult to achieve. We better highlight comparisons to previous methods in several key areas in the Supplemental Table 1. We have also strengthened the Discussion (page 20).

We highlight one line of inquiry enabled by our workflow, a systematic mapping of the cortico-basal- ganglia loops during perceptual decision-making. The striatum is topographically organized. Previous studies examined different subregions of the striatum in different perceptual decision behaviors, making comparisons across studies difficult. The striatum in the mouse brain is ~21.5 mm3 in size (Allen reference brain, (Wang, et al, Cell 2020)). Optogenetic experiments using optical fibers manipulate activity near the fiber tip (approximately 1 mm3). A systematic survey of different striatal domains’ involvement in specific behaviors is currently difficult. In our workflow, individual striatal subregions (~1 mm3, Figure 8) could be rapidly screened through parallel testing. At moderate throughput (15 mice / 2 months), a screen that tiles the entire striatum could be completed in under 12 months with little human effort. To illustrate its feasibility, we tested 3 subregions in the striatum previously implicated in different types of perceptual decision behaviors (Yartsev et al, eLife 2018; Sippy et al, Neuron 2015; Znamenskiy & Zador, Nature 2013), including an additional region in the posterior striatum that do not receive ALM and S1 inputs. The results revealed a hotspot in the dorsolateral striatum that biased tactile-guided decision-making (Figure 8). Our approach thus opens the door to rapid screening of the striatal domains during complex operant behaviors.

Moreover, by eliminating human intervention, automated training allows quantitative assaying of task learning (Figure 4). Home-cage testing also exposes behavioral signatures of motivation in self-initiated behavior (Figure 6). These observations suggest additional opportunities for inquires of goal-directed behaviors in the context of home-cage testing.

Reviewer #3:

In this study, Hao et al. developed an automatized operant box to perform decision-making tasks and optogenetic perturbations without requiring the experimenter's manipulation. For this aim, mice learn to head-fix and to perform a task by themselves. The optogenetic experiment using red-shifted opsins allows manipulation of circuits without the need of an implanted optical fiber. The automation of behavioral tasks in home cages (isolated rodents or in groups) is an intense area of research in neuroscience. The possibility of coupling home cage behavioral analysis with optogenetic manipulation and with complex tasks that require precise positioning of the animal for controlled stimulations (vibrating stimulation, visual …..) is thus of great interest and I commend the authors for their comprehensive dissection of the automated behavioral training setup. Some clarification, reporting of additional behavioral measures and refinement of analyses could improve the impact of this work.

1) The first part of the paper nicely describes the experimental procedure to automate such a complex task. The procedure is very well described, the important points (e.g. the possibility for the animal to disengage…) are properly highlighted, and the online site allows to download the plans and 3D descriptions of the tools and the procedures. The authors compare task learning in automated versus manual training and show that there are overall very few differences. Whisker trimming reduces performance, indicating that animal used information to make the choice. This part of the work is already impressive. Apart from that, the authors do not consider in their description what could be an essential aspect of experiments in a home-cage, i.e the control of the motivation to perform the task. Mice perform the task (here, engage in the head fixation to obtained reward) when they wish and thus, compared with the manual training, there is no explicit control of the animal motivation. This could have consequence on i) the inter-fixation intervals that become an element of the decision and ii) questioned whether the commitment to the task is always motivated by drinking, or whether there is also a commitment to explore, or to check… This could impact the success in the task (e.g. if the animal is not motivated by water, it can explore…). Adding data analyses (information about the daily water consumption, are the inter-fixation intervals correlated with the success or failure in the last trial …) and even short discussion or introduction of these aspects (see for example Timberlake et al, JEAB 1987 or Rowland et al 2008, Physiol behavior for distinction between close and open economies paradigm) could strengthened the behavioral description.

We thank the reviewer for these suggestions. We performed additional analyses to examine these issues which led us to include a new section of Results in the revised manuscript (page 13-14 and Figure 6).

We have added a new Figure 6 showing water consumption and body weight information in home-cage testing. At steady state, a mouse typically consumed ~1mL of water daily (~400 rewarded trials) while maintaining stable body weight. This amount of water consumption was similar to mice engaged in daily manual experiments (Guo et al, Plos ONE 2014). The number of head-fixations per day was correlated with body weight (Figure 6). Since body weight reflects prior water consumption, this indicates different levels of motivation due to thirst, which drives engagement in the task.

We also examined the inter-fixation-interval. Interestingly, the inter-fixation-interval after an error (which led to no reward) was significantly longer than following a correct trial (Figure 6E). This is inconsistent with error from exploration. Rather it likely reflects a loss of motivation after an error, perhaps due to the loss of an expected reward. We suspect that error trials violated the mice’s expectation of reward, and therefore discouraged the mice, leading to a loss in motivation. Consistent with this interpretation, we also found a significant increase in inter-fixation-intervals shortly after a sensorimotor contingency reversal (Figure 6F), coinciding with an increase in error rate due to the rule change.

Despite these changes in motivation to engage in the task, the choice behavior in the task was similar. In highly trained mice, task performance was stable despite the body weight change (Figure 6D). Logistic regression analysis of the choice behavior shows that mice maintained the same strategy in their choice behavior (Figure 6G).

2) In the second part of the work, the authors focus on the description of choice behavior. To characterize it, the authors used a logistic model to predict choices. They suggest that at the beginning of the task the animals biased their current choice by their last choice (parameter A1) and that once the task is learned they alternate according to the current stimulation (parameters S0). The model was a logistic function of the weighted sum of several behavioral and task variables and has 19 parameters (the ß parameters). If the animal only used these two informations, can a model that only takes into account A1 and S0 reproduce the data? If not, this certainly indicates that other informations (even distributed) are necessary; and also indicates individual strategies. Finally, analyses are made by considering trials as a discrete chain (trial n, n+1…). However, the self-head-fixed methodology causes the trials to be organized with more or less time between successive trials depending on motivation (see above). Again, do the authors note differences in performance according to the timing between trials? Could it be a variable in the model?

We thank the reviewer for these great suggestions. We tested a model that included only choice history A1, tactile stimulus S0, and a constant bias term (β0). This 3-parameter model performed as well as the full model in predicting choice. This indicates that other factors do not contribute significantly to the choice behavior. We have included this result in the revised Figure 4C.

We next examined whether inter-fixation-interval (i.e. the time elapsed between head-fixations and presumably the motivation to engage in the task) could impact mice’s choice behavior. There are multiple ways inter-fixation-interval could be incorporated into the logistic regression model. For example, it could be modeled as an explicit variable that biases left/right choice, or modulations on existing regressors (i.e. a gain variable that modulates the contribution of specific regressors). Each approach requires assumptions about how motivation affects the behavioral strategy of the mice. Instead, as a first order analysis, we examined whether the logistic regression model could predict choice equally well in trials following short vs. long inter-fixation-intervals. Our logic is that if mice adapted different strategies in different motivational states (reflected in short vs. long inter-fixation- intervals), the predictive power of the model would differ between these conditions. We fit the logistic regression model using trials in their natural sequential order (regardless of the inter-fixation-intervals). The model was then used to predict choice on independent trials. Trials were then sorted by the preceding inter-fixation-intervals. Prediction performance was calculated separately for trials following short vs. long inter-fixation-intervals. We did not find a significant difference in the model prediction performance. The result was similar in early and late stages of task learning (Figure 6G), even though mice used distinct strategies during these periods (Figure 4). These results suggest consistent strategies in the choice behavior. We have included this analysis in the new Figure 6.

3) The third part described optogenetic manipulations. It is clear that group sizes are small. Nevertheless, if the objective was to show that the method works, the results are convincing. Some experimental details and in particular the choice of the statistical procedure need clarification.

We have improved the presentation and clarified experimental details of the task, hypotheses for targeting specific brain regions, and statistical procedures.

Reviewer #3 (Public Review):

In this study, Hao et al. developed an automatized operant box to perform decision-making tasks and optogenetic perturbations without requiring the experimenter's manipulation. For this aim, mice learn to head-fix and to perform a task by themselves. The optogenetic experiment using red-shifted opsins allows manipulation of circuits without the need of an implanted optical fiber. The automation of behavioral tasks in home cages (isolated rodents or in groups) is an intense area of research in neuroscience. The possibility of coupling home cage behavioral analysis with optogenetic manipulation and with complex tasks that require precise positioning of the animal for controlled stimulations (vibrating stimulation, visual .....) is thus of great interest and I commend the authors for their comprehensive dissection of the automated behavioral training setup. Some clarification, reporting of additional behavioral measures and refinement of analyses could improve the impact of this work.

1) The first part of the paper nicely describes the experimental procedure to automate such a complex task. The procedure is very well described, the important points (e.g. the possibility for the animal to disengage...) are properly highlighted, and the online site allows to download the plans and 3D descriptions of the tools and the procedures. The authors compare task learning in automated versus manual training and show that there are overall very few differences. Whisker trimming reduces performance, indicating that animal used information to make the choice. This part of the work is already impressive. Apart from that, the authors do not consider in their description what could be an essential aspect of experiments in a home-cage, i.e the control of the motivation to perform the task. Mice perform the task (here, engage in the head fixation to obtained reward) when they wish and thus, compared with the manual training, there is no explicit control of the animal motivation. This could have consequence on i) the inter-fixation intervals that become an element of the decision and ii) questioned whether the commitment to the task is always motivated by drinking, or whether there is also a commitment to explore, or to check... This could impact the success in the task (e.g. if the animal is not motivated by water, it can explore...). Adding data analyses (information about the daily water consumption, are the inter-fixation intervals correlated with the success or failure in the last trial ...) and even short discussion or introduction of these aspects (see for example Timberlake et al, JEAB 1987 or Rowland et al 2008, Physiol behavior for distinction between close and open economies paradigm) could strengthened the behavioral description.

2) In the second part of the work, the authors focus on the description of choice behavior. To characterize it, the authors used a logistic model to predict choices. They suggest that at the beginning of the task the animals biased their current choice by their last choice (parameter A1) and that once the task is learned they alternate according to the current stimulation (parameters S0). The model was a logistic function of the weighted sum of several behavioral and task variables and has 19 parameters (the ß parameters). If the animal only used these two informations, can a model that only takes into account A1 and S0 reproduce the data? If not, this certainly indicates that other informations (even distributed) are necessary; and also indicates individual strategies. Finally, analyses are made by considering trials as a discrete chain (trial n, n+1...). However, the self-head-fixed methodology causes the trials to be organized with more or less time between successive trials depending on motivation (see above). Again, do the authors note differences in performance according to the timing between trials? Could it be a variable in the model?

3) The third part described optogenetic manipulations. It is clear that group sizes are small. Nevertheless, if the objective was to show that the method works, the results are convincing. Some experimental details and in particular the choice of the statistical procedure need clarification.

Reviewer #2 (Public Review):

Hao and colleagues developed an automatic system for high-throughput behavioral and optogenetic experiments for mice in home cage settings. The system includes a voluntary head-fixation apparatus and integrated fiber-free optogenetic capabilities. The authors describe in detail the design of the system and the stages for successful automatic training. They perform proof-of-concept experiments to validate their system. The experiments are technically solid and I am convinced that their system will be of interest to some laboratories that perform similar experiments. Despite the large variety of similar automated systems out there, this one may prove to become a popular design.

The weak side of the work is that it is not particularly novel scientifically. The system is complex but there it is not an innovative technology. The body of the study has too many technical details as if it is a Methodological section of a regular manuscript. There are bits of interesting information scattered around the paper (like the insights about the strategy mice use, which stem from the regression analysis), but these are not developed into any coherent direction that answers outstanding questions. The potential advantages of this system compared to other systems is marginal. In my eyes, the fact that manual training is so similar to the automatic one is not only a positive point. Rather, it signifies that the differences are mainly quantitative (e.g. # of mice a lab can train per day, etc). Thus, even as a methods paper, the lack of qualitative difference between this and other methods weakens it as a potential substrate for novel findings.