Observation: Noticed a letter being written which discuss the waterways on a Friday morning

Interpretation: How Lewis or Clark describe their journey exploring the rivers and observing the trees as well.

I think kids do better in school when they can focus, get along with others, and aren’t weighed down by problems at home. For children from low-income families, outside factors can make it really hard to thrive. Things like family stress, lack of access to healthcare, or emotional struggles can slow down their physical and mental development. When kids are distracted by issues like financial strain or tension at home, it affects not only their academic performance but also their overall well-being.

This shows how greatly family income and socioeconomic background affect school results. From access to better schools to lots of resources for extracurricular activities, Alexander's family wealth and suburban surroundings give him major benefits that help him succeed in his academics and in life. Anthony's family, on the other hand, battles lower educational achievement and financial uncertainty, which reduces chances for academic excellence and parental participation in his schooling. The disparity in family structure is also quite important since Alexander gains from the presence of both parents, which not only raises household income but also improves the availability of parental time and support.

This stands out to me so much because of how parenting is reflected based on everything that happens in life. In a household with constant worries and stress the parent tends to reflect those worries on their kids. It is either shown through a parents anger and always responding as if they're annoyed, yelling, and just anxious, which that tends to affect their kids behavior in school or wherever they may be. We look at things different when it comes to spending money.

Observation: The heaviness that the slaves felt from being in slavery and wanting justice and being shown mercy and wanted to ask Thomas Jefferson Questions.

Interpretation:This sentence in the letter was describing the emotion that the anonymous slave felt and also others that was slaves which was feeling burden and wanting to seek justice while also want to experience mercy.

• 2015 -> Volkswagen selling diesel cars as environmentally friendly and fuel efficient which caused high levels of air pollution

• Weiwei incident

• Unlike theft, robbery involves stealing a property while a victim is involved, but the distinction is left to the police

• Fraud example -> Scam emails that asks for our personal information

• Another example of fraud is ponzi's scam

• Wayfair was accused of smuggling children and selling them on the market, but it's not grounded in credible evidence

• 2020

• Happened for the protection of sex workers, but forced them to go to underground sex-work

• Prevented easier screener in part of the sex worker

• Eg. Chemical dependency, grooming through a false sense of love and obligation, etc.

• Exploits the human need for connection, even when there's exploitation and abuse involved

• Stockholm syndrome -> Captive develops a bond with their captor as a defense mechanism under extreme defense

• Trafficking involves trafficking transporting people to another country without their consent, for the purpose of forcing them to be involved in sex trades or in forced labour

• What, why, and how are the three elements of human trafficking

• Smuggling involves transporting people to another country consensuallly, often by means of coercion and deception.

• Smuggling victims are often involved due to unsafe environments in their home country and seeking a better help elsewhere

• Engaging in collective behavior for mutual benefit
• Eg. Challenger Explosion in 1986 ->

• Perpetrated by the government, individuals in the government, or an individual in the government
• Eg. War crimes Real life example: Residential schools of Canada
• State crimes occur mostly for money

• Business-relate crime such as stealing money from employer -> Embezzlement
• Eg. Ponzi scheme -> Taking advantage of people in what seems to be a legitimate business

• Group of organization or individuals in that organization engages in illegal acts
• Eg. bribing, insider trading
• Real life example: Jordan Belfort who commited stock price fixing

This has always been an issue that has been known. In this diverse society the teachers themselves pick and choose who they want to help out and see succeed. Big public schools have always to be known to segregate people of color because they do not think they have the same capacity as thers to be able to educationally succeed. On the other hand, in a school very diverse majority of the time we all tend to push very hard but because of the feeling of being let down and not having support we tend to slack off and not do the full potential.

why did charles martel stoped the muslims from conquering france?

this is crazy! Charleigh practice annotate

woop woop it works

wonder why they think its non science based?

Case: patient is named case #2, male

Disease Assertion: UCD/OTCD

Family Info:

Case Presenting HPOs: Hyperammonemia (HP:0001987), oriticaciduria (HP:0003218), low plasma citrulline (HP:0003572), neonatal onset(HP:0003623), Hyperglutaminemia (HP:0003217)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: GDNA was isolated from lymphocytes. To examine the small mutations in the coding region of the OTC gene, all 10 exons and their flanking intron regions were amplified using PCR, and the nucleotide sequences of the amplified products were determined. To determine the intron 5 sequence of case 2, PCR was performed using primers OTCex5F and OTCint5R, and primers OTCint5F and OTCex6R (Table 1, Fig. 3). The amplified products were subcloned into the pT7 vector and the inserted DNA was sequenced using an automated DNA sequencer. Allopurinol test

Supplemental Data: TABLE 1, Notes:

Variant: NM_000531.6: c.540+265G>A

ClinVarID: NA

CAID: CA658658977

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Reviewer #2 (Public review):

Goldstein et al. provide a thorough characterization of the interaction of attention and eye movement planning. These processes have been thought to be intertwined since at least the development of the Premotor Theory of Attention in 1987, and their relationship has been a continual source of debate and research for decades. Here, Goldstein et al. capitalize on their novel urgent saccade task to dissociate the effects of endogenous and exogenous attention on saccades towards and away from the cue. They find that attention and eye movements are, to some extent, linked to one another but that this link is transient and depends on the nature of the task. A primary strength of the work is that the researchers are able to carefully measure the time course of the interaction between attention and eye movements in various well-controlled experimental conditions. As a result, the behavioral interplay of two forms of attention (endogenous and exogenous) are illustrated at the level of tens of milliseconds as they interact with the planning and execution of saccades towards and away from the cued location. Overall, the results allow the authors to make meaningful claims about the time course of visual behavior, attention, and the potential neural mechanisms at a timescale relevant to everyday human behavior.

Reviewer #3 (Public review):

The present study used an experimental procedure involving time-pressure for responding, in order to uncover how the control of saccades by exogenous and endogenous attention unfolds over time. The findings of the study indicate that saccade planning is influenced by the locus of endogenous attention, but that this influence was short-lasting and could be overcome quickly. Taken together, the present findings reveal new dynamics between endogenous attention and eye movement control and lead the way for studying them using experiments under time-pressure.

The results achieved by the present study advance our understanding of vision, eye movements, and their control by brain mechanisms for attention. In addition, they demonstrate how tasks involving time-pressure can be used to study the dynamics of cognitive processes. Therefore, the present study seems highly important not only for vision science, but also for psychology, (cognitive) neuroscience, and related research fields in general.

I think the authors' addressed all of the reviewers' points successfully and in detail, so that I don't have any further suggestions or comments.

Author response:

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

Reviewer #1 (Public Review):

The main research question could be defined more clearly. In the abstract and at some points throughout the manuscript, the authors indicate that the main purpose of the study was to assess whether the allocation of endogenous attention requires saccade planning [e.g., ll.3-5 or ll.247-248]. While the data show a coupling between endogenous attention and saccades, they do not point to a specific direction of this coupling (i.e., whether endogenous attention is necessary to successfully execute a saccade plan or whether a saccade plan necessarily accompanies endogenous attention).

Thanks for the suggestion. We have modified the text in the abstract and at various points in the text to make it more clear that the study investigates the relationship between attention and saccades in one particular direction, first attentional deployment and then saccade planning.

Some of the analyses were performed only on subgroups of the participants. The reporting of these subgroup analyses is transparent and data from all participants are reported in the supplementary figures. Still, these subgroup analyses may make the data appear more consistent, compared to when data is considered across all participants. For instance, the exogenous capture in Experiments 1 and 2 appears much weaker in Figure 2 (subgroup) than Figure S3 (all participants). Moreover, because different subgroups were used for different analyses, it is often difficult to follow and evaluate the results. For instance, the tachometric curves in Figure 2 (see also Figure 3 and 4) show no motor bias towards the cue (i.e., performance was at ~50% for rPTs <75 ms). I assume that the subsequent analyses of the motor bias were based on a very different subgroup. In fact, based on Figure S2, it seems that the motor bias was predominantly seen in the unreliable participants. Therefore, I often found the figures that were based on data across all participants (Figures 7 and S3) more informative to evaluate the overall pattern of results.

Indeed, our intent was to dissociate the effects on saccade bias and timing as clearly as possible, even if that meant having to parse the data into subgroups of participants for different analyses. We do think conceptually this is the better strategy, because the bias and timing effects were distinct and not strongly correlated with specific participants or task variants. For instance, the unreliable participants were somewhat more consistently biased in the same direction, but the reliable participants also showed substantial biases, so the difference in magnitude was relatively modest. This can be more easily appreciated now that the reliable and unreliable participants are indicated in Figures 3 and 5. The impact of the bias is also discussed further in the last paragraphs of the Results, which note that the bias was not a reliable predictor of overall success during informed choices.

Reviewer #3 (Public Review):

(1) In this experimental paradigm, participants must decide where to saccade based on the color of the cue in the visual periphery (they should have made a prosaccade toward a green cue and an antisaccade away from a magenta cue). Thus, irrespective of whether the cue signaled that a prosaccade or an antisaccade was to be made, the identity of the cue was always essential for the task (as the authors explain on p. 5, lines 129-138). Also, the location where the cue appeared was blocked, and thus known to the participants in advance, so that endogenous attention could be directed to the cue at the beginning of a trial (e.g., p. 5, lines 129-132). These aspects of the experimental paradigm differ from the classic prosaccade/antisaccade paradigm (e.g. Antoniades et al., 2013, Vision Research). In the classic paradigm, the identity of the cues does not have to be distinguished to solve the task, since there is only one stimulus that should be looked at (prosaccade) or away from (antisaccade), and whether a prosaccade or antisaccade was required is constant across a block of trials. Thus, in contrast to the present paradigm, in the classic paradigm, the participants do not know where the cue is about to appear, but they know whether to perform a prosaccade or an antisaccade based on the location of the cue.

The present paradigm keeps the location of the cue constant in a block of trials by intention, because this ensures that endogenous attention is allocated to its location and is not overpowered by the exogenous capture of attention that would happen when a single stimulus appeared abruptly in the visual field. Thus, the reason for keeping the location of the cue constant seems convincing. However, I wondered what consequences the constant location would have for the task representations that persist across the task and govern how attention is allocated. In the classic paradigm, there is always a single stimulus that captures attention exogenously (as it appears abruptly). In a prosaccade block, participants can prioritize the visual transient caused by the stimulus, and follow it with a saccade to its coordinates. In an antisaccade block, following the transient with a saccade would always be wrong, so that participants could try to suppress the attention capture by the transient, and base their saccade on the coordinates of the opposite location. Thus, in prosaccade and antisaccade blocks, the task representations controlling how visual transients are processed to perform the task differ. In the present task, prosaccades and antisaccades cannot be distinguished by the visual transients. Thus, such a situation could favor endogenous attention and increase its influence on saccade planning, even though saccade planning under more naturalistic conditions would be dominated by visual transients. I suggest discussing how this (and vice versa the emphasis on visual transients in the classic paradigm) could affect the generality of the presented findings (e.g., how does this relate to the interpretation that saccade plans are obligatorily coupled to endogenous attention? See, Results, p. 10, lines 306-308, see also Deubel & Schneider, 1996, Vision Research).

Great discussion point. There are indeed many ways to set up an experiment where one must either look to a relevant cue or look away from it. Furthermore, it is also possible to arrange an experiment where the behavior is essentially identical to that in the classic antisaccade task without ever introducing the idea of looking away from something (Oor et al., 2023). More important than the specific task instructions or the structure of the event sequence, we think the fundamental factors that determine behavior in all of these cases are the magnitudes of the resulting exogenous and endogenous signals, and whether they are aligned or misaligned. Under urgent conditions, consideration of these elements and their relevant time scales explains behavior in a wide variety of tasks (see Salinas and Stanford, 2021). Furthermore, a recent study (Zhu et al., 2024) showed that the activation patterns of neurons in monkey prefrontal cortex during the antisaccade task can be accurately predicted from their stimulus- and saccade-related responses during a simpler task (a memory guided saccade task). This lends credence to the idea that, at the circuit level, the qualities that are critical for target selection and oculomotor performance are the relative strengths of the exogenous and endogenous signals, and their alignment in space and time. If we understand what those signals are, then it no longer matters how they were generated. The Discussion now includes a paragraph on this issue.

(2) Discussion (p. 16, lines 472-475): The authors suppose that "It is as if the exogenous response was automatically followed by a motor bias in the opposite direction. Perhaps the oculomotor circuitry is such that an exogenous signal can rapidly trigger a saccade, but if it does not, then the corresponding motor plan is rapidly suppressed regardless of anything else.". I think this interesting point should be discussed in more detail. Could it also be that instead of suppression, other currently active motor plans were enhanced? Would this involve attention? Some attention models assume that attention works by distributing available (neuronal) processing resources (e.g., Desimone & Duncan, 1995, Annual Review of Neuroscience; Bundesen, 1990, Psychological Review; Bundesen et al., 2005, Psychological Review) so that the information receiving the largest share of resources results in perception and is used for action, but this happens without the active suppression of information.

The rebound seen after the exogenously driven changes is certainly interesting, and we agree that it could involve not only the suppression of a specific motor plan but also enhancement of another (opposite) plan. However, we think that, given the lack of prior data with the requisite temporal precision, further elaboration of this point would just be too speculative in the context of the point that we are trying to make, which is simply that the underlying choice dynamics are more rapid and intricate than is generally appreciated.

(3) Methods, p. 19, lines 593-596: It is reported that saccades were scored based on their direction. I think more information should be provided to understand which eye movements entered the analysis. Was there a criterion for saccade amplitude? I think it would be very helpful to provide data on the distributions of saccade amplitudes or on their accuracy (e.g. average distance from target) or reliability (e.g. standard deviation of landing points). Also, it is reported that some data was excluded from the analysis, and I suggest reporting how much of the data was excluded. Was the exclusion of the data related to whether participants were "reliable" or "unreliable" performers?

The reported results are based on all saccades (detected according to a velocity threshold) that were produced after the go signal and in a predominantly horizontal direction (within ± 60° of the cue or non-cue), which were the vast majority (> 99%). Indeed, most saccades were directed to the choice targets, with 95% of them within ± 14.2° of the horizontal plane. The excluded (non-scored) trials were primarily fixation breaks plus a small fraction of trials with blinks, which compromised saccade determination. There was no explicit amplitude criterion; applying one (for instance, excluding any saccades with amplitude < 2°) produced minimal changes to the data. Overall, saccade amplitudes were distributed unimodally with a median of 7.7° and a 95% confidence interval of [3.7°, 9.7°], whereas the choice targets were located at ± 8° horizontally. This is now reported in the Methods.

As far as data exclusion, analyses were based on urgent trials (gap > 0); non-urgent (gap < 0) trials were excluded from calculation of the tachometric curves simply because they might correspond to a slightly different regime (go signal after cue onset) and to long processing times in the asymptotic range (rPT in 200–300 ms) or beyond, which are not as informative. However, including them made no appreciable difference to the results. No data were excluded based on participant performance or identity; all psychometric analyses were carried out after the selection of trials based on the scoring criteria described above. This is now stated in the Methods.

(4) Results, p. 9, lines 262-266: Some data analyses are performed on a subset of participants that met certain performance criteria. The reasons for this data selection seem convincing (e.g. to ensure empirical curves were not flat, line 264). Nevertheless, I suggest to explain and justify this step in more detail. In addition, if not all participants achieved an acceptable performance and data quality, this could also speak to the experimental task and its difficulty. Thus, I suggest discussing the potential implications of this, in particular, how this could affect the studied mechanisms, and whether it could limit the presented findings to a special group within the studied population.

The ideal (i.e., best) analysis for determining the cost of an antisaccade for each individual participant (Fig. 4c) was based on curve fitting and required task performance to rise consistently above chance at long rPTs in both pro and anti trials. This is why the mentioned conditions on the fits were imposed. This is now explained in the text. This ideal analysis was not viable for all tachometric curves not necessarily because of task difficulty but also because of high variability or high bias in a particular experiment/condition. It is true that the task was somewhat difficult, but this manifested in various ways across the dataset, so attempting to draw a clean-cut classification of participants based on “difficulty” may not be easy or all that informative (as can be gleaned from Fig. S1). There simply was a range of success levels, as one might expect from any task that requires some nontrivial cognitive processing. Also note that no participants were excluded flat out from analysis. Thus, at the mentioned point in the text, we simply note that a complementary analysis is presented later that includes all participants and all conditions and provides a highly consistent result (namely, Fig. 7e). Then, in the last section of the Results, where Fig. 7 is presented, we point out that there is considerable variance in performance at long rPTs, and that it relates to both the bias and the difficulty of the task across participants.   

Reviewer #1 (Recommendations For The Authors):

(1) I have some questions related to the initial motor bias:

a) Based on Figure S3, which shows the tachometric curves using data from all participants, there only seems to be a systematic motor bias in Experiments 1 and 3 but no bias in Experiments 2 and 4. It is unclear to me why this is different from the data shown in Figure 7.

For the bars in Fig. 7, accuracy (% correct) was computed for each participant and then averaged across participants, whereas for the data in Fig. S3, trials were first pooled across participants and then accuracy was computed for each rPT bin. The different averaging methods produce slightly different results because some participants had more trials in the guessing range than others, and different biases.  

b) Based on Figure 7 (and Figure S3), there was no motor bias in Experiment 4. Based on the correlations between motor bias and time difference between pro and antisaccades, I would expect that the rise points between pro and antisaccades would be more similar in this Experiment. Was this the case?

No. Figs. 3c and S3d show that the rise times of pro and anti trials for Experiment 4 still differ by about 30 ms (around the 75% correct mark), and the rest of the panels in those figures show that the difference is similar for all experiments. What happens is that Figs. 7 and S3 show that on average the bias is zero for Experiment 4, but that does not mean that the average difference in rise times is zero because there is an offset in the data (correlation is not the same as regression). The most relevant evidence is in Fig. 6c, which shows that, for an overall bias of zero, one would still expect a positive difference in rise times of about 25–30 ms. This figure now includes a regression line, and the corresponding text now explains the relationship between bias and rise times more clearly. Thanks for asking; this is an important point that was not sufficiently elaborated before.

c) If I understand correctly, the initial motor bias was predominantly observed in participants who were classified as 'unreliable performers' (comparing Figure S2 and Figure 2). Was there a correlation between the motor bias and overall success in the task? In other words: Was a strong motor bias generally disadvantageous?

Good question. Participants classified as ‘unreliable’ were somewhat more consistently biased in the same direction than those classified as ‘reliable’, but the distinction in magnitude was not large. This can be better appreciated now in Fig. 5 by noting the mix of black (reliable) and gray labels (unreliable) along the x axes. The unreliable participants were also, by definition, less accurate in their asymptotic performance in at least one experiment (Fig. S1). In general, however, this classification was used simply to distinguish more clearly the two main effects in the data (timing cost and bias). In fact, the motor bias was not a reliable predictor of performance during informed choices: across all participants, the mean accuracy in the asymptotic range (rPT > 200 ms) had a weak, non-significant correlation with the bias (ρ = ‒0.07, p = 0.7). So, no, the motor bias did not incur an obvious disadvantage in terms of overall success in the task. Its more relevant effect was the asymmetry in performance that it promoted between pro- and antisaccade trials (Fig. 6c). This is now explained at the end of the Results.

(2) One of the key analyses of the current study is the comparison of the rPT required to make informed pro and antisaccades (ll.246 ff). I think it would be informative for readers to see the results of this analysis separately for all four experiments. For instance, based on Figure 4a and b, it looks like the rise points were actually very similar between pro and antisaccades in Experiment 1.

We agree that the ideal analysis would be to compute the performance rise point for pro- and antisaccade curves for each experiment and each participant, but as is now noted in the text, this requires a steady and substantial rise in the tachometric curve, which is not always obtained at such a fine-grained level; the underlying variability can be glimpsed from the individual points in Fig. 7a, b. Indeed, in Fig. 4a, b the mean difference between pro and anti rise points appears small for Experiment 1 — but note that the two panels include data from only partially overlapping sets of participants; the figure legend now makes this more clear. Again, this is because the required fitting procedure was not always reliable in both conditions (pro and anti) for a given subject in a given experiment. Thus, panels a and b cannot be directly compared. The key results are those in Fig. 4c, which compare the rise points in the two conditions for the same participants (11 of them, for which both rise points could be reliably determined). In that case the mean difference is evident, and the individual effect consistent for 9 of the 11 participants (as now noted).

A similar comparison for Experiments 1 or 2 individually would include fewer data points and lose statistical power. However, on average, the results for Experiments 1 and 2 (separately) were indeed very similar; in both cases, the comparison between pro and anti curves pooled across the same qualifying participants as in Fig. 4c produced results that were nearly identical to those of Fig. 4d (as can be inferred from Fig. 2a, b). Furthermore, results for the four individual experiments pooled across all participants are presented in Figure S3, which shows delayed rises in antisaccade performance consistent with the single participant data (Fig. 4c).

(3) Figure 3: It would be helpful to indicate the reliable performers that were used for Figure 3a in the bar plots in Figure 3b. Same for Figures 3c and d.

Done. Thanks for the suggestion.

(4) Introduction: The literature on the link between covert attention and directional biases in microsaccades seems relevant in the context of the current study (e.g., Hafed et al., 2002, Vision Res; Engbert & Kliegl, 2003, Vision Res; Willett & Mayo, 2023, Proc Natl Acad Sci USA).

Yes, thanks for the suggestion. The introduction now mentions the link between attentional allocation and microsaccade production.

(5) ll.395ff & Figure 7f: Please clarify whether data were pooled across all four experiments for this analysis.

Yes, the data were pooled, but a positive trend was observed for each of the four experiments individually. This is now stated.

(6) ll.432-433: There is evidence that the attentional locus and the actual saccade endpoint can also be dissociated (e.g., Wollenberg et al., 2018, PLoS Biol; Hanning et al., 2019, Proc Natl Acad Sci USA).

True. We have rephrased accordingly. Thanks for the correction.

(7) ll.438-440: This sentence is difficult to parse.

Fixed.

Reviewer #2 (Recommendations For The Authors):

The manuscript is well-written and compelling. The biggest issue for me was keeping track of the specifics of the individual experiments. I think some small efforts to reinforce those details along the way would help the reader. For example, in the Figure 3 figure legend, I found the parenthetical phrase "high luminence cue, low luminence non-cue)" immensely helpful. It would be helpful and trivial to add the corresponding phrase after "Experiment 4" in the same legend.

Thanks for the suggestion. Legends and/or labels have been expanded accordingly in this and other figures.

Line 314: "..had any effect on performance,..." Should there be a callout to Figure 2 here?

Done.

It wasn't clear to me why the specific high and low luminance values (48 and 0.25) were chosen. I assume there was at least some quick perceptual assessment. If that's the case or if the values were taken from prior work, please include that information.

Done.

Reviewer #3 (Recommendations For The Authors):

Minor points. Please note that the comments made in the public review above are not repeated here.

(1) Introduction, p. 2, lines 41-45: It is mentioned that the effects of covert attention or a saccade can be quite distinct. I suggest specifying in what way.

Done.

(2) Introduction, p. 2, lines 46-47: It is said that the relation between attention and saccade planning was still uncertain and then it is stressed that this was the case for more natural viewing conditions. However, the discussed literature and the experimental approach of the current study still rely on experimental paradigms that are far from natural viewing conditions. Thus, I suggest either discussing the link between these paradigms and natural viewing in more detail or leaving out the reference to natural viewing at this point (I think the latter suggestion would fit the present paper best).

We followed the latter suggestion.

(3) Introduction (e.g. p. 3, lines 55-58): The authors discuss the effects that sustaining fixation might have on attention and eye movements. Recently, it has been found that maintaining fixation can ameliorate cognitive conflicts that involve spatial attention (Krause & Poth, 2023, iScience). It seems interesting to include this finding in the discussion, because it supports the authors' view that it is necessary to study fixation and eye movements rather than eye movements alone to uncover their interplay with attention and decision-making.

Thanks for the reference. The reported finding is certainly interesting, but we find it somewhat tangential to the specific point we make about strong fixation constraints — which is that they suppress internally driven motor activity, including biases, that are highly informative of the relationship between attention and saccade planning (lines 466‒472, 541‒561). Whether fixation state has other subtle consequences for cognitive control is an intriguing, important issue, for sure. But we would rather maintain the readers’ focus on the reasons why less restrictive fixation requirements are relevant for understanding the deployment of attention.

(4) Results, p. 9, lines 264-266: It is reported that "The rise points were statistically the same across experiments for both prosaccades (p=0.08, n=10, permutation test)...", but the p-value seems quite close to significance. I suggest mentioning this and phrasing the sentence a bit more carefully.

We now refer to the rise points as “similar”.

(5) Figure 7 a-d: It might help readers who first skim through the figures before reading the text to use other labels for the bins on the x-axis that spell out the name of the phase in the trial. It might also help to visualize the bins on the plot of a tachymetric function (in this case, changing the labels could be unnecessary).

Thanks for the suggestion. We added an insert to the figure to indicate the correspondence between labels and time bins more intuitively.

(6) Methods, p. 18, lines 566-567: On some trials, participants received an auditory beep as a feedback stimulus. As this could induce a burst of arousal, I wondered how it affected the subsequent trials.

This is an interesting issue to ponder. We agree that, in principle, the beep could have an impact on arousal. However, what exactly would be predicted as a consequence? The absence of a beep is meant to increase the urgency of the participant, so some effect of the beep event on RT would be expected anyway as per task instructions. Thus, it is unclear whether an arousal contribution could be isolated from other confounds. That said, three observations suggest that, at most, an independent arousal effect would be very small. First, we have performed multisensory experiments (unpublished) with auditory and visual stimuli, and have found that it is difficult to obtain a measurable effect of sound on an urgent visual choice task unless the experimental conditions are particularly conducive; namely, when the visual stimuli are dim and the sound is loud and lateralized. None of these conditions applies to the standard feedback beep. Second, because most trials are on time, the meaningful feedback signal is conveyed by the absence of the beep. But this signal to alter behavior (i.e., respond sooner) has zero intensity and is therefore unlikely to trigger a strong exogenous, automatic response. Finally, in our data, we can parse the trials that followed a beep (the majority) from those that did not (a minority). In doing so, we found no differences with respect to perceptual performance; only minor differences in RT that were identical for pro- and antisaccade trials. All this suggests to us that it is very unlikely that the feedback alters arousal significantly on specific trials, somehow impacting the tachometric curve (a contribution to general arousal across blocks or sessions is possible, of course, but would be of little consequence to the aims of the study).

(7) Methods, p. 18, lines 574-577: I suggest referring to the colors or the conditions in the text as it was done in the experiments, just to prevent readers being confused before reading the methods.

We appreciate the thought, but think that the study is easier to understand by pretending, initially, that the color assignments were fixed. This is a harmless simplification. Mentioning the actual color assignments early on would be potentially more confusing and make the description of the task longer and more contrived.

(8) Methods, p. 18, Table 1: Given that the authors had a spectrophotometer, I suggest providing (approximate) measurements for the stimulus colors in addition to the luminance (i.e. not just RGB values).

Unfortunately, we have since switched the monitor in our setup, so we don’t have the exact color measurements for the stimuli used at the time. We will keep the suggestion in mind for future studies though.

References

Oor EE, Stanford TR, Salinas E (2023) Stimulus salience conflicts and colludes with endogenous goals during urgent choices. iScience 26:106253.

Salinas E, Stanford TR (2021) Under time pressure, the exogenous modulation of saccade plans is ubiquitous, intricate, and lawful. Curr Opin Neurobiol 70:154-162.

Zhu J, Zhou XM, Constantinidis C, Salinas E, Stanford TR (2024) Parallel signatures of cognitive maturation in primate antisaccade performance and prefrontal activity. iScience.  doi: https://doi.org/10.1016/j.isci.2024.110488.

I think the difficuility that they had were the seasons changing, the weather, or some of the work had to be done physically.

intersting to show how other technologies are based from older developments from other regions.

Interesting that backpropagation really is just the chain rule over and over again. I wonder why it took us so long to realize the strengths of DL.

It's interesting that you do not see many Taylor series approaches in the context of deep neural networks. The idea that you can create nonlinear decision boundaries with nested perceptrons, but we do not use Taylor series, which appear powerful enough to do that too is interesting. Is it computational limits?

like observing the glacier collapse/melting at certain point that no one would expect to happen

key points

It's quite like the process of scholars doing research since we all are supposed to conduct our own research on the basis of preexisting findings. That's exactly why we want to do literature review.

issue of bias

Welcome back.

In this lesson, I'll be talking about Network Address Translation, or NAT, a process of giving a private resource outgoing only access to the internet.

And a NAT gateway is the AWS implementation that's available within WPC.

There's quite a bit of theory to cover, so let's get started.

So what is NAT?

Well, it stands for Network Address Translation.

This is one of those terms which means more than people think that it does.

In a strict sense, it's a set of different processes which can adjust ID packets by changing their source or destination IP addresses.

Now, you've seen a form of this already.

The internet gateway actually performs a type of NAT known as static NAT.

It's how a resource can be allocated with a public IP version for address, and then when the packets of data leave those resources and pass through the internet gateway, it adjusts the source IP address on the packet from the private address to the public, and then sends the packet on, and then when the packet returns, it adjusts the destination address from the public IP address to the original private address.

That's called static NAT, and that's how the internet gateway implements public IP version for addressing.

Now, what most people think of when they think of NAT is a subset of NAT called IP Masquerading.

And IP Masquerading hides a whole private side IP block behind a single public IP.

So rather than the one private IP to one public IP process that the internet gateway does, NAT is many private IPs to one single IP.

And this technique is popular because IP version 4 addresses are running out.

The public address space is rapidly becoming exhausted.

IP Masquerading, or what we'll refer to for the rest of this lesson as NAT, gives a whole private range of IP addresses outgoing only access to the public internet and the AWS public zone.

I've highlighted outgoing because that's the most important part, because many private IPs use a single public IP.

Incoming access doesn't work.

Private devices that use NAT can initiate outgoing connections to internet or AWS public space services, and those connections can receive response data, but you cannot initiate connections from the public internet to these private IP addresses when NAT is used.

It doesn't work that way.

Now, AWS has two ways that it can provide NAT services.

Historically, you could use an EC2 instance configured to provide NAT, but it's also a managed service, the NAT gateway, which you can provision in the VPC to provide the same functionality.

So let's look at how this works architecturally.

This is a simplified version of the Animals for Life architecture that we've been using so far.

On the left is an application tier subnet in blue, and it's using the IP range 10.16.32.0/20.

So this is a private only subnet.

Inside it are three instances, I01, which is using the IP 10.16.32.10, I02, which is using 32.20, and I03, which is using 32.30.

These IP addresses are private, so they're not publicly routable.

They cannot communicate with the public internet or the AWS public zone services.

These addresses cannot be routed across a public style network.

Now, if we wanted this to be allowed, if we wanted these instances to perform certain activities using public networking, for example, software updates, how would we do it?

Well, we could make the subnet's public in the same way that we've done with the public subnets or the web subnets, but we might not want to do that architecturally.

With this multi-tier architecture that we're implementing together, part of the design logic is to have tiers which aren't public and aren't accessible from the public internet.

Now, we could also host some kind of software update server inside the VPC, and some businesses choose to do that.

Some businesses run Windows update services, all Linux update services inside their private network, but that comes with an admin overhead.

NAT offers us a third option, and it works really well in this style of situation.

We provision a NAT gateway into a public subnet, and remember, the public subnet allows us to use public IP addresses.

The public subnet has a route table attached to it, which provides default IP version 4 routes pointing at the internet gateway.

So, because the NAT gateway is located in this public web subnet, it has a public IP which is routable across the public internet, so it's now able to send data out and get data back in return.

Now, the private subnet where the instances are located can also have its own route table, and this route table can be different than the public subnet route table.

So, we could configure it so that the route table that's on the application subnet has a default IP version 4 route, but this time, instead of pointing at the internet gateway, like the web subnet users, we configure this private route table so that it points at the NAT gateway.

This means when those instances are sending any data to any IP addresses that do not belong inside the VPC, by default, this default route will be used, and that traffic will get sent to the NAT gateway.

So, let's have a look at how this packet flow works.

Let's simulate the flow packets from one of the private instances and see what the NAT gateway actually does.

So, first, instance 1 generates some data.

Let's assume that it's looking for software updates.

So, this packet has a source IP address of instance 1's private IP and a destination of 1.3.3.7.

For this example, let's assume that that's a software update server.

Now, because we have this default route on the route table of the application subnet, that packet is routed through to the NAT gateway.

The NAT gateway makes a record of the data packet.

It stores the destination that the packet is for, the source address of the instance sending it, and other details which help it identify the specific communication in future.

Remember, multiple instances can be communicating at once, and for each instance, it could be having multiple conversations with different public internet hosts.

So, the NAT gateway needs to be able to uniquely identify those.

So, it records the IP addresses involved, the source and destination, the port numbers, everything it needs, into a translation table.

So, the NAT gateway maintains something called a translation table which records all of this information.

And then, it adjusts the packet to the one that's been sent by the instance, and it changes the source address of this IP packet to be its own source address.

Now, if this NAT appliance were anywhere for AWS, what it would do right now is adjust the packet with a public routable address. - Hi. - Let's do this directly.

But remember, all the inside of the IPC really has directly attached to it a public IP version 4 address.

That's what the internet gateway does.

So, the NAT gateway, because it's in the web subnet, it has a default route, and this default route points at the internet gateway.

And so, the packet is moved from the NAT gateway to the internet gateway by the IPC router.

At this point, the internet gateway knows that this packet is from the NAT gateway.

It knows that the NAT gateway has a public IP version 4 address associated with it, and so, it modifies the packet to have a source address of the NAT gateway's public address, and it sends it on its way.

The NAT gateway's job is to allow multiple private IP addresses to masquerade behind the IP address that it has.

That's where the term IP masquerading comes from.

That's why it's more accurate.

So, the NAT gateway takes all of the incoming packets from all of the instances that it's managing, and it records all the information about the communication.

It takes those packets, it changes the source address from being those instances to its own IP address, its own external-facing IP address.

If it was outside AWS, this would be a public address directly.

That's how your internet router works for your home network.

All of the devices internally on your network talk out using one external IP address, your home router uses NAT.

But because it's in AWS, it doesn't have directly attached a real public IP.

The internet gateway translates from its IP address to the associated public one.

So, that's how the flow works.

If you need to give an instance its own public IP version for address, then only the internet gateway is required.

If you want to give private instances outgoing access to the internet and the AWS public services such as S3, then you need both the NAT gateway to do this many-to-one translation and the internet gateway to translate from the IP of the NAT gateway to a real public IP version for address.

Now, let's quickly run through some of the key facts for the NAT gateway product that you'll be implementing in the next demo lesson.

First, and I hope this is logical for you by now, it needs to run from a public subnet because it needs to be able to be assigned a public IP version for IP address for itself.

So, to deploy a NAT gateway, you already need your VPC in a position where it has public subnets.

And for that, you need an internet gateway, subnets configured to allocate public IP version for addresses and default routes for those subnets pointing at the internet gateway.

Now, a NAT gateway actually uses a special type of public IP version for address that we haven't covered yet called an elastic IP.

For now, just know that these are IP version for addresses, which is static.

They don't change.

These IP addresses are allocated to your account in a region and they can be used for whatever you want until you reallocate them.

And NAT gateways use these elastic IPs, the one service which utilizes elastic IPs.

Now, they're talking about elastic IPs later on in the course.

Now, NAT gateways are an AZ resilient service.

If you read the AWS documentation, you might get the impression that they're fully resilient in a region like an internet gateway.

They're not, they're resilient in the AZ that they're in.

So they can recover from hardware failure inside an AZ.

But if an AZ entirely fails, then the NAT gateway will also fail.

For a fully region resilient service, so to mirror the high availability provided by an internet gateway, then you need to deploy one NAT gateway in each AZ that you're using in the VPC and then have a route table for private subnets in that availability zone, pointing at the NAT gateway also in that availability zone.

So for every availability zone that you use, you need one NAT gateway and one route table pointing at that NAT gateway.

Now, they aren't super expensive, but it can get costly if you have lots of availability zones, which is why it's important to always think about your VPC design.

Now, NAT gateways are a managed service.

You deploy them and AWS handle everything else.

They can scale to 45 gigabits per second in bandwidth and you can always deploy multiple NAT gateways and split your subnets across multiple provision products.

If you need more bandwidth, you can just deploy more NAT gateways.

For example, you could split heavy consumers across two different subnets in the same AZ, have two NAT gateways in the same AZ and just route each of those subnets to a different NAT gateway and that would quickly allow you to double your available bandwidth.

With NAT gateways, you'll build based on the number that you have.

So there's a standard hourly charge for running a NAT gateway and this is obviously subject to change in a different region, but it's currently about four cents per hour.

And note, this is actually an hourly charge.

So partial hours are billed as full hours.

And there's also a data processing charge.

So that's the same amount as the hourly charge around four cents currently per gigabyte of processed data.

So you've got this base charge that a NAT gateway consumes while running plus a charge based on the amount of data that you process.

So keep both of those things in mind for any NAT gateway related questions in the exam.

Don't focus on the actual values, just focus on the fact they have two charging elements.

Okay, so this is the end of part one of this lesson.

It's getting a little bit on the long side, and so I wanted to add a break.

It's an opportunity just to take a rest or grab a coffee.

Part two will be continuing immediately from the end of part one.

So go ahead, complete the video, and when you're ready, join me in part two.

Welcome back.

In this lesson I want to talk in detail about security groups within AWS.

These are the second type of security filtering feature commonly used within AWS, the other type being network access control lists which we've previously discussed.

So security groups and knuckles share many broad concepts but the way they operate is very different and it's essential that you understand those differences and the features offered by security groups for both the exam and real-world usage.

So let's just jump in and get started.

In the lesson on network access control lists I explained that they're stateless and by now you know what stateless and stateful mean.

Security groups are stateful, they detect response traffic automatically for a given request and this means that if you allow an inbound or outbound request then the response is automatically allowed.

You don't have to worry about configuring ephemeral ports, it's all handled by the product.

If you have a web server operating on TCP 443 and you want to allow access from the public internet then you'll add an inbound security group rule allowing inbound traffic on TCP 443 and the response which is using ephemeral ports is automatically allowed.

Now security groups do have a major limitation and that's that there is no explicit deny.

You can use them to allow traffic or you can use them to not allow traffic and this is known as an implicit deny.

So if you don't explicitly allow traffic then you're implicitly denying it but you can't and this is important you're unable to explicitly deny traffic using security groups and this means that they can't be used to block specific bad actors.

Imagine you allow all source IP addresses to connect to an instance on port 443 but then you discover a single bad actor is attempting to exploit your web server.

Well you can't use security groups to block that one specific IP address or that one specific range.

If you allow an IP or if you allow an IP range or even if you allow all IP addresses then security groups cannot be used to deny a subset of those and that's why typically you'll use network access control lists in conjunction with security groups where the knuckles are used to add explicit denies.

Now security groups operate above knuckles on the OSI7 layer stack which means that they have more features.

They support IP and side-based rules but they also allow referencing AWS logical resources.

This includes all the security groups and even itself within rules.

I'll be covering exactly how this works on the next few screens.

Just know at this stage that it enables some really advanced functionality.

An important thing to understand is that security groups are not attached to instances nor are they attached to subnet.

They're actually attached to specific elastic network interfaces known as ENIs.

Now even if you see the user interface present this as being able to attach a security group to an instance know that this isn't what happens.

When you attach a security group to an instance what it's actually doing is attaching the security group to the primary network interface of that instance.

So remember security groups are attached to network interfaces that's an important one to remember for the exam.

Now at this point let's step through some of the unique features of security groups and it's probably better to do this visually.

Let's start with a public subnet containing an easy to instance and this instance has an attached primary elastic network interface.

On the right side we have a customer Bob and Bob is accessing the instance using HDTBS so this means TCP but 443.

Conceptually think of security groups as something which surrounds network interfaces in this case the primary interface of the EC2 instance.

Now this is how a typical security group might look.

It has inbound and outbound rules just like a network ACL and this particular example is showing the inbound rules allowing TCP port 443 to connect from any source.

The security group applies to all traffic which enters or leaves the network interface and because they're stateful in this particular case because we've allowed TCP port 443 as the request portion of the communication the corresponding response part the connection from the instance back to Bob is automatically allowed.

Now lastly I'm going to repeat this point several times throughout this lesson.

Security groups cannot explicitly block traffic.

This means with this example if you're allowing 0.0.0.0.0 to access the instance on port TCP port 443 and this means the whole IP version for internet then you can't block anything specific.

Imagine Bob is actually a bad actor.

Well in this situation security groups cannot be used to add protection.

You can't add an explicit deny for Bob's IP address.

That's not something that security groups are capable of.

Okay so that's the basics.

Now let's look at some of the advanced bits of security group functionality.

Security groups are capable of using logical references.

Let's step through how this works with a similar example to the one you just saw.

We start with a VPC containing a public web subnet and a private application subnet.

Inside the web subnet is the Categoram application web instance and inside the app subnet is the back-end application instance.

Both of these are protected by security groups.

We have A4L-web and A4L-app.

Traffic wise we have Bob accessing the web instance over port TCP port 443 and because this is the entry point to the application which logically has other users than just Bob we're allowing TCP port 443 from any IP version for address and this means we have a security group with an inbound rule set which looks like this.

In addition to this front-end traffic the web instance also needs to connect with the application instance and for this example let's say this is using TCP port 1337.

Our application is that good.

So how best to allow this communication?

Well we could just add the IP address of the web instance into the security group of the application instance or if you wanted to allow our application to scale and change IPs then we could add the side arrangers of the subnets instead of IP addresses.

So that's possible but it's not taking advantage of the extra functionality which security groups provide.

What we could do is reference the web security group within the application security group.

So this is an example of the application security group.

Notice that it allows TCP port 1337 inbound but it references as the source a logical resource the security group.

Now using a logical resource reference in this way means that the source reference of the A4L-web security group this actually references anything which has this security group associated with it.

So in this example any instances which have the A4L-web security group attached to them can connect to any instances which have the A4L-web security group attached to them using TCP port 1337.

So in essence this references this.

So this logical reference within the application security group references the web security group and anything which has the web security group attached to it.

Now this means we don't have to worry about IP addresses or side arrangers and it also has another benefit.

It scales really well.

So as additional instances are added to the application subnet and web subnet and as those instances are attached to the relevant security groups they're impacted by this logical referencing allowing anything defined within the security group to apply to any new instances automatically.

Now this is critical to understand so when you reference a security group from another security group what you're actually doing is referencing any resources which have that security group associated with them.

So this substantially reduces the admin overhead when you have multi-tiered applications and it also simplifies security management which means it's prone to less errors.

Now logical references provide even more functionality.

They allow self referencing.

Let's take this as an example a private subnet inside AWS with an ever-changing number of application instances.

Right now it's three but it might be three, thirty or one.

What we can do is create a security group like this.

This one allows incoming communications on port TCP 1337 from the web security group but it also has this rule which is a self-referential rule allowing all traffic.

What this means is that if it's attached to all of the instances then anything with this security group attached can receive communication so all traffic from this security group and this effectively means anything that also has this security group attached to it.

So it allows communications to occur to instances which have it attached from instances which have it attached.

It handles any IP changes automatically which is useful in these instances within an auto scaling group which is provisioning and terminating instances based on load on the system.

It also allows for simplified management of any intra-app communications.

An example of this might be Microsoft the main controllers or managing application high availability within clusters.

So this is everything I wanted to cover about security groups within AWS.

So there's a lot of functionality and intelligence that you gain by using security groups versus network ACLs but it's important that you understand that while network ACLs do allow you to explicitly deny traffic security groups don't and so generally you would use network ACLs to explicitly block any bad actors and use security groups to allow traffic to your VPC based resources.

You do this because security groups are capable of this logical resource referencing and that means AWS logical resources in security groups or even itself to allow this free flow of communications within a security group.

At this point that is everything I wanted to cover in this lesson so go ahead and complete the video and when you're ready I'll look forward to you joining me in the next lesson.

peak literature

Emerson seems like quite the stoic philosopher.

CONNECTION ANNOTATIONS

Just as in the Trott and Lee LLM article, word vectors are described as a matchmaking site for words. AI generated photos could be viewed in a similar way. Not every date will result in a soul mate, therefore every match is not ideal. I often find myself frustrated when typing and my cell phone will decide that it is smarter than me and knows exactly what I am going to say next. I become irate when I hit send before, I realize those changes have been made and I sound as though I only completed my elementary school education. The same can be send for these photos, when an AI model acquires many photos to compile one master image, thought to be exactly what you are looking for, only to discover that AI is so generous, that they have provided the subject of the photo with an extra arm.

1. Similar to how Trott and Lee LLM article phrased AI as being similar to "turning the water on, making sure it's coming out of the right faucet, and when it's not, running around to tighten and loosen all the valves until it does", AI generated photos (7:20) can create biases that then we must run around to resolve. The original information inputted to generate a photo is like the water. Once it is turned on and a photo is generated, but it's not the photo we were expecting, we then have to tweak and edit the information until we receive a desired outcome.

does this make sense to you? Why or why not?

Motivation!

Goals ought to be SMART!

strategic behaviour...

perhaps this is where we might talk about S.M.A.R.T. goals...

one of my favourite definitions of entrepreneurship

the motivating question -- what brings about success?

what's in your toolbox?

d'uh

and intelligence (the successful type!)

!

*

Historical causality- this unique circumstance rose from __ Social causality- A may lead to B or increases the chances of B

in certain contexts we can expect behavior but can never count on it in all instances

follow many different "causal chains" to reach conclusions as opposed to one single cause and affect

probability of human behavior doesn't come from notions of free will as much as it comes from the variety of situations and possibilities that can't always be accounted for in strict categories

maybe Durkheim would differ

eLife Assessment

This technical study presents a novel sampling strategy for detecting synaptic coupling between neurons from dual pipette patch-clamp recordings in acute slices of mammalian brain tissue in vitro. The authors present solid evidence that this strategy, which incorporates automated patch clamp electrode positioning and cleaning for reuse with strategic neuron targeting, has the potential to substantially improve the efficiency of neuronal sampling with paired recordings. This technique and the extensions discussed will be useful for neuroscientists wanting to apply or already conducting automated multi-pipette patch clamp recording electrophysiology experiments in vitro for neuron connectivity analyses.

Reviewer #1 (Public review):

Summary:

In this technical paper, the authors introduce an important variation on the fully automated multi-electrode patch-clamp recording technique for probing synaptic connections that they term "patch-walking". The patch-walking approach involves coordinated pipette route-planning and automated pipette cleaning procedures for pipette reuse to improve recording throughput efficiency, which the authors argue can theoretically yield almost twice the number of connections to be probed by paired recordings on a multi-patch electrophysiology setup for a given number of cells compared to conventional manual patch-clamping approaches used in brain slices in vitro. The authors show convincing results from recordings in mouse in vitro cortical slices, demonstrating the efficient recording of dozens of paired neurons with a dual patch pipette configuration for paired recordings and detection of synaptic connections. This approach will be of interest and valuable to neuroscientists conducting automated multi-patch in vitro electrophysiology experiments and seeking to increase efficiency of neuron connectivity detection while avoiding the more complex recording configurations (e.g., 8 pipette multi-patch recording configurations) used by several laboratories that are not readily implementable by most of the neuroscience community.

Strengths:

(1) The authors introduce the theory and methods and show experimental results for a fully automated electrophysiology dual patch-clamp recording approach with a coordinated patch-clamp pipette route-planning and automated pipette cleaning procedures to "patch-walk" across an in vitro brain slice.

(2) The patch-walking approach offers throughput efficiency improvements over manual patch clamp recording approaches, especially for investigators looking to utilize paired patch electrode recordings in electrophysiology experiments in vitro.

(3) Experimental results are presented from in vitro mouse cortical slices demonstrating the efficiency of recording dozens of paired neurons with a two-patch pipette configuration for paired recordings and detection of synaptic connections, demonstrating the feasibility and efficiency of the patch-walking approach.

(4) The authors suggest extensions of their technique while keeping the number of recording pipettes employed and recording rig complexity low, which are important practical technical considerations for investigators wanting to avoid the more complex recording configurations (e.g., 8-10 pipette multi-patch recording configurations) used by several laboratories that are not readily implementable by most of the neuroscience community.

Reviewer #2 (Public review):

Summary:

In this study, the authors aim to combine automated whole-cell patch clamp recording simultaneously from multiple cells. Using a 2-electrode approach, they are able to sample as many cells (and connections) from one slice, as would be achieved with a more technically demanding and materially expensive 8-electrode patch clamp system. They provide data to show that this approach is able to successfully record from 52% of attempted cells, which was able to detect 3 pairs in 71 screened neurons. The authors state that this is a step forward in our ability to record from randomly connected ensembles of neurons.

Strengths:

The conceptual approach of recording multiple partner cells from another in a step wise manner indeed increases the number of tested connections. An approach that is widely applicable to both automated and manual approaches. Such a method could be adopted for many connectivity studies using dual recording electrodes.

The implementation of automated robotic whole-cell patch-clamp techniques from multiple cells simultaneously is a useful addition to the multiple techniques available to ex vivo slice electrophysiologists.

The approach using 2 electrodes, which are washed between cells is economically favourable, as this reduces equipment costs for recording multiple cells, and limits the wastage of capillary glass that would otherwise be used once.

Weaknesses:

(1) Based on the revised manuscript - a discussion of the implementation of this approach to manual methods is still lacking,

(2) A comparison of measurements shown in Figure 2 to other methods has not been addressed adequately.

(3) The morphological identification of neurons is understandably outside the remit of this project - but should be discussed and/or addressed. It was not suggested to perform detailed anatomical analysis - but to highlight the importance of this, and it should still be discussed

(4) The revised manuscript does not clearly state which cells were included in the analysis as far as I can see - and indeed cells with Access Resistance >40 MOhm appear to still be included in the data.

Reviewer #3 (Public review):

Summary:

In this manuscript, Yip and colleagues incorporated the pipette cleaning technique into their existing dual-patch robotic system, "the PatcherBot", to allow sequential patching of more cells for synaptic connection detection in living brain slices. During dual-patching, instead of retracting all two electrodes after each recording attempt, the system cleaned just one of the electrodes and reused it to obtain another recording while maintaining the other. With one new patch clamp recording attempt, new connections can be probed. By placing one pipette in front of the other in this way, one can "walk" across the tissue, termed "patch-walking." This application could allow for probing additional neurons to test the connectivity using the same pipette in the same preparation.

Strengths:

Compared to regular dual-patch recordings, this new approach could allow for probing more possible connections in brain slices with dual-patch recordings, thus having the potential to improve the efficiency of identifying synaptic connections

Weaknesses:

While this new approach offers the potential to increase efficiency, it has several limitations that could curtail its widespread use.

Loss of Morphological Information: Unlike traditional multi-patch recording, this approach likely loses all detailed morphology of each recorded neuron. This loss is significant because morphology can be crucial for cell type verification and understanding connectivity patterns by morphological cell type.

Spatial Restrictions: The robotic system appears primarily suited to probing connections between neurons with greater spatial separation (~100µm ISD). This means it may not reliably detect connections between neurons in close proximity, a potential drawback given that the connectivity is much higher between spatially close neurons. This limitation could help explain the low connectivity rate (5%) reported in the study.

Limited Applicability: While the approach might be valuable in specific research contexts, its overall applicability seems limited. It's important to consider scenarios where the trade-off between efficiency and specific questions that are asked.<br /> Scalability Challenges: Scaling this method beyond a two-pipette setup may be difficult. Additional pipettes would introduce significant technical and logistical complexities.

Author response:

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

We thank the reviewers and editors for insightful feedback on how we could improve the manuscript. We have revised the manuscript and addressed the points raised.

Regarding the technical issues raised about the quality of patch clamp recordings (Reviewer 2), we acknowledge that the upper limit of the access resistance cutoff should be lower and that the accepted change should be 10-20%. To this end, we have revised the manuscript to more accurately detail the quality metrics used. The access resistance for the neurons in paired recordings were below 40 MΩ (similar to the metric used by Kolb et al. 2019), and if the access changed above 50 MΩ, we stopped recording from that neuron. Furthermore, the inclusion of neurons in the histogram with access resistance above 50 MΩ was to highlight the total number of neurons patched but not necessarily used in paired recordings. As this was done with an automated robotic system, the neurons would still undergo an initial voltage clamp and current clamp protocol before the pipette would release the neuron and patch another cell. To the point of Reviewer 2, this patch-walk protocol could also be alternatively implemented using manual recording approaches and this point has been included in the revised manuscript.

Regarding the spatial restrictions (Reviewer 3), we agree that the average intersomatic distance is higher than ideal. This was likely due to failed patch attempts; for instance, if one pipette successfully achieved whole cell, and the other pipette had several sequential failed patch attempts, the intersomatic distance (ISD) would increase with each failed attempt due to the user selected index of cells. Ideally, the pipettes would be walking across a slice with low ISD if the whole-cell success rate was closer to 100%. To overcome this challenge in future work, automated cell identification and tracking could enable the path planning to be continuously updated after each patch attempt. Given the whole-cell success rate efficiency for a given electrophysiologist, we believe that the automated robot could be improved in later versions to include routeplanning algorithms to minimize the distance between neurons. Alternatively, this patch-walk system could also be integrated to improve connectivity yields for manual recording approaches as well.

For the point raised about morphological identification, we believe that while important, morphological identification is out of the scope for this project. Future work will include neuronal reconstruction. Regarding the other points, we will amend the manuscript to highlight other key metrics such as maximum time we could hold a neuron under the whole-cell configuration. Additionally, we agree with Reviewer 3 that some of the current language may cause confusion, and we will amend it accordingly.

To all the reviewers, thank you for your time, understanding, and the opportunity to improve our manuscript.

now that's interesting (working potentially against self-interest)

...

!

broader context (calling for more specific details)

!!

another opportunity for me to share a Viktor Frankl quote: "Don't aim at success ... For success, like happiness, cannot be pursued; it must ensue"

handy reminder...

a moral agenda, without moralizing ... hah

this idea of "happiness gone awry" is tied more to hedonism, but a psychopath could certainly get a sense of fulfilment from inflicting pain on others.

wow!

so it's not that a healthy attitude/mindset automatically confers health benefits - it may simply mean you avail yourself more of the services that correlate with health benefits...

still sounding awfully parochial

yep

interesting...

two mutually related concerns!

(entrepreneurial intention begets entrepreneurial action (or not, if entrepreneurial stress intervenes), but eventual action begets such stress inevitably, which begets other health concerns, which might affect future entrepreneurial intent/action...)

a sobering reminder

!!

!!

eudaemonia in the everyday! Super important. We don't just feel fulfilled when accomplishing significant tasks.

How does eudaimonic well-being manifest in your mundane experiences?

fascinating observation - different kinds of stressors produce different responses...

mutual constitution!

well-being (Eudaemonia) moderates continued perseverance (not just starting ventures)

a feeling of fulfilment (sense of accomplishment across the different components of well-being) leads to other positive feelings (positive affect / happiness), which in turn leads to more effort, engagement, success, etc...

proposed explanation...

compared to "regular" folk...

Broaden and build:

While negative emotions have been associated with survival and protection in response to a threatening situation, positive emotions have been related to the ability to explore the environment, to be open to new information, to create and build new resources. According to this theory, positive emotions broaden the scope of attention, enabling flexible thinking. This in turn facilitates the development of new skills, networks, and capacities that are essential to adaptively handle a stressful event.

In the long-term, people who experience more positive emotions are more satisfied with their lives, build more positive relationships with partners, get better jobs, or even have better health.

so ... these sort of findings aren't limited to necessity entrepreneurs ...

can't discount well-being. Mental health is important for physical (and financial) health!

interesting ... the passion part of grit...

aha! Not all stress is bad. And stress can be mitigated by one's well-being (it actually has to be).

link back to grit!

ouch!

A thought provoking distinction...

not very encouraging. Desperation and such conditions lead to stress (often of the chronic, not the acute type). And this stress has negative implications on one's health and well-being.

...

seems kind of obvious...

The same way money doesn't buy happiness, autonomy doesn't mean independence. Interesting...

!!

Exactly!

!

useful distinction!

Dang! So social entrepreneurship can help change folks' economic status, but not necessarily their subjective well-being! It might actually worsen it ... hmmm.... even though it increases their autonomy.

What's your take on this?

What's your take on this?

so, less money can = more problems!

ooh - key distinction.

4th theme

the distinction, crystallized again...

5th theme...

5 key concerns...

2nd theme

3rd theme

Autonomy = BOTHJ motive and an aspect of well-being. Interesting!

like EQ ... and a growth mindset...

if you're happier, and functioning well, you're likely to be healthier too...

...

food for thought...

...

wow - awesome link to previous weeks' material...

ok...

self-acceptance!

??

...

So many good Frankl quotes!

honesty is the best policy (especially with oneself!) As it can prevent you from getting in over your head...

(this is related to the question I asked at the end of our last class -- do you envy people who lead an easy life? Where do they find meaning?)

Purpose in life!

positive relations!

Personal growth!

AUTONOMY

a most useful question...

human fulfillment doesn't mean one is a fully functioning human...

(Makes me think of teleology and acorns...)

Environmental mastery (tied to social settings and social skills in emotional intelligence...)

(mostly) the focus of the other article this week...

getting very philosophical here (but context is good)

which one is more important? Can you say this? Can one become what one is and live up to one's true potential without knowing oneself? Hmmmmm......

I love how this definition is prefaced with "kind of"

all righty then...

telling us what is to follow...

!!

Completely different focus! Well, not "completely" distinct, but shifting focus away from happiness that comes from achieving things to happiness from achieving purpose ...

hedonism - often associated with the pursuit of pleasure.

This article doesn't focus on it a lot (since it emphasizes the other main aspect of well-being), but I'd like to just highlight the connection between hedonism and happiness a little bit here by emphasizing the concept of the "hedonic treadmill".

The hedonic treadmill is a metaphor for the human tendency to pursue one pleasure after another. That's because the surge of happiness that's felt after a positive event is likely to return to a steady personal baseline over time.

So, it looks like this:

This is why I was so interested in the discussion we had at the end of the last class (which we'll continue this week) about money and happiness. Mo money, mo problems. We desire more riches, but we're never satisfied ...

Which is one of the reasons we need to look beyond hedonic well-being and explore eudaimonic well-being!

so, this is going to cover a lot of ground!

as we know, trying to find our purpose or constantly "develop"/optimize/life-hack/actualize/etc, isn't always a recipe for happiness (or even "success")

ok - we were already introduced to this distinction in the last article - we're going to get into it more here!

ok - so we're not going to do that here...

ok - we are familiar with this rhetorical contextextualizing... (it's the last half of our whole course)

oof

!

huh

curious - given the next statement...

makes sense - entrepreneurs value independent thinking...

a fabulously evocative image.

background...

love it -- one can't ignore self-interest!

interesting...

narrowing the focus...

ok

!

aha - morality again (but distinct from that of social entrepreneurs) ...

let's take the road less travelled!

https://www.derstandard.at/story/3000000239670/ex-google-ceo-will-mehr-ki-datenzentren-statt-klimaziele?ref=niewidget

.

certainly linked to Csikszentmihalyi's view on happiness!

ooh! I really like this link to the previous week on perseverance and grit...

meaningful and purposeful work can also help constitute a happy and successful life (!)

unless you're into stats and quantitative measurement, you can skip over these details ... but the social significance rather than the statistical significance of their findings is still trenchant!

like the lingo!

all right -- leading us into the next article!

generally accepted as entrepreneurs are assumed to get more meaning and purpose from their work than folk who work for someone/thing else other than themselves...

yep

9 factors to measure what matters ...

double yep

interesting

operationalizing flow, or success, or productivity is difficult enough -- but operationalizing subjective well-being ... sheesh! I think, for our purposes, it's best to interrogate all of these things and not pinpoint them or measure them but assess how they function within a mix of variables to try and reflect on our own intersection with these concepts. We're not trying to "hack" happiness here, but a bit more accurately map its coordinates so that we can find ourselves there when our internal GPS might be going a little funky...

• check it out in the quiz section this week...

ok! -- there are a number of different tests to capture your capacity for "flow" states...

wow! When listed in this way, it seems as though most workplaces or types of work might not encourage group productivity. How many of these apply to your work?

*

**

so, meaning matters more than money when it comes to motivation...

making a difference (for others...) or making a difference for oneself (being personally fulfilled)...

summing up perceptions of success

ooh - a link to next week...

makes sense!

not as far as the marketplace is concerned...

meaning matters, wherever you find it...

ok...

ok

profits and power are important, but are byproducts of passion (!)

hmmm

*

the testable proposition...

yowza!

ok!

can you relate this to your own experiences?

remember what I said in my note about happiness and flow in the seminar prompt in week 7 (creative/cultural entrepreneurs)