The captain is mute, also I dont think its ever voluntary to be mentaly ill

(like 15% for example), even though the

the sensor will only see down to

eLife Assessment

This study provides an important re-evaluation of modality-specific information processing in the thalamus of trained mice. Using an elegant task design that probes competing tactile and visual stimuli, the authors present compelling evidence that behavioral training reshapes the sensitivity of higher-order thalamic nuclei. Despite the powerful task design and the significance of the main findings, the origin of the cross-modal responses remains an open question and requires future investigation.

Reviewer #1 (Public review):

Petty and Bruno investigate how response characteristics in the higher-order thalamic nuclei POm (typically somatosensory) and LP (typically visual) change when a stimulus (whisker air puff or visual drifting grating) of one or the other modality is conditioned to a reward. Using a two-step training procedure, they developed an elegant paradigm, where the distractor stimulus is completely uninformative about the reward, which is reflected in licking behavior of trained mice. While the animals seem to take on to the tactile stimulus more readily, they can also associate reward with the visual stimulus, ignoring tactile stimuli. In trained mice, the authors recorded single unit responses in both POm and LP while presenting the same stimuli. The authors first focused on POm recordings, finding that in animals with tactile conditioning POm units specifically responded to the air puff stimulus but not the visual grating. Unexpectedly, in visually conditioned animals, POm units also responded to the visual grating, suggesting that the responses are not modality-specific but more related to behavioral relevance. These effects seem not not be homogeneously distributed across POm, whereas lateral units maintain tactile specificity and medial units respond more flexibly. The authors further ask if the unexpected cross-modal responses might result from behavioral activity signatures. By regressing behavior-coupled activity out of the responses, they show that late activity indeed can be related to whisking, licking and pupil size measures. However, cross-modal short latency responses are not clearly related to animal behavior. Finally, LP neurons also seem to change their modality-specificity dependent on conditioning, whereas tactile responses are attenuated in LP if the animal is conditioned to visual stimuli.

The authors make a compelling case that POm neurons are less modality specific than typically assumed. The training paradigm, employed methods and analyses are to the point, well supporting the conclusions. The findings importantly widen our understanding of higher-order thalamus processing features with flexibility to encode multiple modalities and behavioral relevance. The results raise many important questions on the brain-wide representation of conditioned stimuli. E.g. how specific are the responses to the conditioned stimuli? Are thalamic cross-modal neurons recruited for the specific conditioned stimulus or do their responses reflect a more global shift of attention from one modality to another? Are these cross-modal responses tracking global arousal/attention features, or actually encoding a different stimulus?

The authors clarified a number of points in the updated version of the manuscript and expanded analyses and methods descriptions, which substantially improved the paper. The different time periods around the stimuli are more clearly assigned now and make the conclusions stronger.

Especially the discussion is now well rounded and addresses the major points.

To ask if the cross-modal activity is in some way functional for task performance I would like to see if (population) activity in the classical vs. cross-modal nucleus is predictive of lick latency or frequency on a trial-to-trial basis.

I accept that the authors cannot differentiate between bottom-up "raw" sensory responses and top-down context/attention/etc signals and thus support the decision to restrict the analyses to either the likely sensory early part following stimulus onset or the (as shown here mostly movement-driven) offset period after cessation of the stimulus. However, the composite responses over different stimuli and conditioning types seem triphasic to me. I find the "ongoing" activity differences (~100-2000 ms) depending on conditioning type quite interesting and would welcome a more specific discussion on the different response periods.

Overall a very elegant and well-presented study.

Reviewer #2 (Public review):

This manuscript by Petty and Bruno delves into the still poorly understood role of higher-order thalamic nuclei in the encoding of sensory information by examining the activity in the Pom and LP cells in mice performing an associative learning task. They developed an elegant paradigm in which they conditioned head-fixed mice to attend to a stimulus of one sensory modality (visual or tactile) and ignore a second stimulus of the other modality. They recorded simultaneously from POm and LP, using 64-channels electrode arrays, to reveal the context-dependency of the firing activity of cells in higher-order thalamic nuclei. They concluded that behavioral training reshapes activity in these secondary thalamic nuclei. The authors brought new analyses and figures which greatly improve their manuscript and support their conclusion. The manuscript benefits now from a better communication about both the methodology and the results. I have no more major concerns, but I feel that the readability of the manuscript could be improved with the following revisions.

Strengths

The authors developed an original and elegant paradigm in which they conditioned head-fixed mice to attend to a stimulus of one sensory modality, either visual or tactile and ignore a second stimulus of the other modality. As a tactile stimulus, they applied gentle air puffs on the distal part of the vibrissae, ensuring that the stimulus was innocuous and therefore none aversive which is crucial in their study.

It is commonly viewed that first-order thalamus performs filtering and re-encoding of the sensory flow; in contrast the computations taking place in high-order nuclei are poorly understood. They may contribute to cognitive functions. By integrating top-down control, high-order nuclei may participate in generating update models of the environment based on sensory activity; how this can take place is a key question that Petty and Bruno addressed in the present study.

Weaknesses

(1) It's difficult when reading the text to understand which results were quantified and which were not, in part because mean data as well as (s.e.m. or S.D.) do not appear either in the main text nor in the legends of the figures. Only vague and unquantified data are given in the main text. I understand that the authors may want to make the main text less heavy, but having these data fully written somewhere (i.e., main text, summary table, figure legends) rather than having to estimate through looking at a graph (especially when the data are constraint in the first 20% of the graph (Figure 4c)), would greatly improve the text's clarity and precision.

For instance, Line #173, "At the population level, POm cells in both conditioning groups had a peak of activity 40ms after air puff onset (Figure 4a)." Is this 40 ms a result of quantified data, then a s.e.m. would be informative, or a reading measurement on the Figure 4a graphs? As it stands, it is too vague a value.

(2) The authors give clearer definition of what they analyzed, which greatly improved the readability of the manuscript. The clarity of the manuscript could still be improved by solving remaining ambiguities about sensory- versus non-sensory-responses to the applied stimuli throughout the manuscript, in order to better convey the authors' conclusion that behavioral training reshapes activity in these secondary thalamic nuclei which then may participate in generating update models of the context in which the animal is performing the task.

Line #24 in the abstract "In mice trained to respond to tactile stimuli and ignore visual stimuli, POm was robustly activated by touch and largely unresponsive to visual stimuli". The abstract would better reflect the manuscript conclusions indicating that POm was robustly activated during tactile stimuli.

(3) The new analysis of the "early" responses in Pom cells pointed out, Line #173, that "At the population level, POm cells in both conditioning groups had a peak of activity 40ms after air puff onset (Figure 4a)." Previous works cited by the authors, Diamond et al. (1992), described tactile responses in Pom cells at 15-20ms latency which were suppressed by the barrel cortex inactivation.

The 40ms-latency responses described in this manuscript therefore do not fit with "purely sensory" and barely with S1-feedbacks, as proposed on line #168 "Such responses could be "purely sensory" (i.e. driven by ascending brainstem inputs)" or line #334 "It is likely that the observed activity in lateral dorsal POm is driven by true whisker responses in SpVi and S1."

In the same way, Line #315 "we observed POm cells that responded to the onset of the air puff in both conditioning groups". This conclusion should be dampened, to better fit the results, by "we observed POm cells that responded 40 ms after the onset of the air puff in both conditioning groups."

Reviewer #3 (Public review):

Petty and Bruno ask whether activity in secondary thalamic nuclei depends on the behavioral relevance of stimulus modality. They recorded from POm and LP, but the weight of the paper is skewed toward POm. They use two cohorts of mice (N=11 and 12), recorded in both nuclei using multi-electrode arrays, while being trained to lick to either a tactile stimulus (air puff against whiskers, first cohort) or a visual stimulus (drifting grating, second cohort), and ignore the respective other. They find that both nuclei, while primarily responsive to their 'home' modality, are more responsive to the relevant modality (i.e. the modality predicting reward).

Strengths:

The paper asks an important question, it is timely and is very well executed. The behavioral method using a delayed lick index (excluding impulsive responses) is well worked out. Electrophysiology methods are state-of-the-art with information about spike quality in Fig. S1. The main result is novel and important, convincingly conveying the point that encoding of secondary thalamic nuclei is flexible and clearly includes aspects of the behavioral relevance of a stimulus. The paper explores the mapping of responses within POm, pointing to a complex functional structure, something that has been reported/suggested in earlier studies.

Weaknesses:

Coding: It does not become clear to which aspect of the task POm/LP are responding. There is a motor-related response (whisking, licking, pupil), which, however, after regressing it out leaves a remaining response that the authors speculate could be sensory.

Learning: The paper talks a lot about 'learning', although it is only indirectly addressed. The authors use two differently (over-)trained mice cohorts rather than studying e.g. a rule switch in one and the same mouse, which would allow to directly assess whether it is the same neurons that undergo rule-dependent encoding

Mapping: The authors present electrode tracks with marked selectivity indices of recordings in POm and LP. This is a great start, but to finally understand the functional composition of POm and LP, a more detailed and systematic mapping effort is needed in the future.

Author response:

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

Reviewer #1 (Public Review): 

Petty and Bruno investigate how response characteristics in the higher-order thalamic nuclei POm (typically somatosensory) and LP (typically visual) change when a stimulus (whisker air puff or visual drifting grating) of one or the other modality is conditioned to a reward. Using a two-step training procedure, they developed an elegant paradigm, where the distractor stimulus is completely uninformative about the reward, which is reflected in the licking behavior of trained mice. While the animals seem to take on to the tactile stimulus more readily, they can also associate the reward with the visual stimulus, ignoring tactile stimuli. In trained mice, the authors recorded single-unit responses in both POm and LP while presenting the same stimuli. The authors first focused on POm recordings, finding that in animals with tactile conditioning POm units specifically responded to the air puff stimulus but not the visual grating. Unexpectedly, in visually conditioned animals, POm units also responded to the visual grating, suggesting that the responses are not modality-specific but more related to behavioral relevance. These effects seem not be homogeneously distributed across POm, whereas lateral units maintain tactile specificity and medial units respond more flexibly. The authors further ask if the unexpected cross-modal responses might result from behavioral activity signatures. By regressing behavior-coupled activity out of the responses, they show that late activity indeed can be related to whisking, licking, and pupil size measures. However, cross-modal short latency responses are not clearly related to animal behavior. Finally, LP neurons also seem to change their modality-specificity dependent on conditioning, whereas tactile responses are attenuated in LP if the animal is conditioned to visual stimuli.

The authors make a compelling case that POm neurons are less modality-specific than typically assumed. The training paradigm, employed methods, and analyses are mostly to the point, well supporting the conclusions. The findings importantly widen our understanding of higher-order thalamus processing features with the flexibility to encode multiple modalities and behavioral relevance. The results raise many important questions on the brain-wide representation of conditioned stimuli. E.g. how specific are the responses to the conditioned stimuli? Are thalamic cross-modal neurons recruited for the specific conditioned stimulus or do their responses reflect a more global shift of attention from one modality to another? 

To elaborate on higher-order thalamic activity in relationship to conditioned behavior, a trialby-trial analysis would be very useful. Is neuronal activity predictive of licking and at which relative timing? 

To elaborate on the relationship between neuronal activity and licking, we have created a new supplementary figure (Figure S1), where we present the lick latency of each mouse on the day of recording. We also perform more in-depth analysis of neural activity that occurs before lick onset, which is presented in a new main figure (new Figure 4). 

Furthermore, I wonder why the (in my mind) major and from the data obvious take-away, "POm neurons respond more strongly to visual stimuli if visually conditioned", is not directly tested in the summary statistics in Figure 3h.

We have added a summary statistic to Figure 3h and to the Results section (lines 156-157) comparing the drifting grating responses in visually and tactilely conditioned mice.  

The remaining early visual responses in POm in visually conditioned mice after removing behavior-linked activity are very convincing (Figure 5d). It would help, however, to see a representation of this on a single-neuron basis side-by-side. Are individual neurons just coupled to behavior while others are independent, or is behaviorally coupled activity a homogeneous effect on all neurons on top of sensory activity?

In lieu of a new figure, we have performed a new analysis of individual neurons to classify them as “stimulus tuned” and/or “movement tuned.” We find that nearly all POm cells encode movement and arousal regardless of whether they also respond to stimuli. This is presented in the Results under the heading “POm correlates with arousal and movement regardless of conditioning” (Lines 219-231).

The conclusions on flexible response characteristics in LP in general are less strongly supported than those in POm. First, the differentiation between POm and LP relies heavily on the histological alignment of labeled probe depth and recording channel, possibly allowing for wrong assignment. 

We appreciate the importance in differentiating between POm, LP, and surrounding regions to accurately assign a putative cell to a brain region. The method we employed (aligning an electrode track to a common reference atlas) is widely used in rodent neuroscience, especially in regions like POm and LP which are difficult to differentiate molecularly (for example, see Sibille, Nature Communications, 2022; and Schröder, Neuron, 2020). 

Furthermore, it seems surprising, but is not discussed, that putative LP neurons have such strong responses to the air puff stimuli, in both conditioning cases. In tactile conditioning, LP air puff responses seem to be even faster and stronger than POm. In visual conditioning, drifting grating responses paradoxically seem to be later than in tactile conditioning (Fig S2e). These differences in response changes between POm and LP should be discussed in more detail and statements of "similar phenomena" in POm and LP (abstract) should be qualified.  

We have further developed our analysis and discussion of LP activity. Our analysis of LP stimulus response latencies are now presented in greater detail in Figure S3, and we have expanded the results section accordingly (lines 266-275). We have also expanded the discussion section to both address these new analyses and speculate on what might drive these surprising “tactile responses” in LP.

Reviewer #2 (Public Review): 

Summary  

This manuscript by Petty and Bruno delves into the still poorly understood role of higherorder thalamic nuclei in the encoding of sensory information by examining the activity in the Pom and LP cells in mice performing an associative learning task. They developed an elegant paradigm in which they conditioned head-fixed mice to attend to a stimulus of one sensory modality (visual or tactile) and ignore a second stimulus of the other modality. They recorded simultaneously from POm and LP, using 64-channel electrode arrays, to reveal the contextdependency of the firing activity of cells in higher-order thalamic nuclei. They concluded that behavioral training reshapes activity in these secondary thalamic nuclei. I have no major concerns with the manuscript's conclusions, but some important methodological details are lacking and I feel the manuscript could be improved with the following revisions.

Strengths 

The authors developed an original and elegant paradigm in which they conditioned headfixed mice to attend to a stimulus of one sensory modality, either visual or tactile, and ignore a second stimulus of the other modality. As a tactile stimulus, they applied gentle air puffs on the distal part of the vibrissae, ensuring that the stimulus was innocuous and therefore none aversive which is crucial in their study. 

It is commonly viewed that the first-order thalamus performs filtering and re-encoding of the sensory flow; in contrast, the computations taking place in high-order nuclei are poorly understood. They may contribute to cognitive functions. By integrating top-down control, high-order nuclei may participate in generating updated models of the environment based on sensory activity; how this can take place is a key question that Petty and Bruno addressed in the present study.

Weaknesses  

(1) Overall, methods, results, and discussion, involving sensory responses, especially for the Pom, are confusing. I have the feeling that throughout the manuscript, the authors are dealing with the sensory and non-sensory aspects of the modulation of the firing activity in the Pom and LP, without a clear definition of what they examined. Making subsections in the results, or a better naming of what is analyzed could convey the authors' message in a clearer way, e.g., baseline, stim-on, reward.  

We thank Reviewer 2 for this suggestion. We have adjusted the language throughout the paper to more clearly state which portions of a given trial we analyzed. We now consistently refer to “baseline,” “stimulus onset,” and “stimulus offset” periods. 

In line #502 in Methods, the authors defined "Sensory Responses. We examined each cell's putative sensory response by comparing its firing rate during a "stimulus period" to its baseline firing rate. We first excluded overlapping stimuli, defined as any stimulus occurring within 6 seconds of a stimulus of a different type. We then counted the number of spikes that occurred within 1 second prior to the onset of each stimulus (baseline period) and within one second of the stimulus onset (stimulus period). The period within +/-50ms of the stimulus was considered ambiguous and excluded from analysis." 

Considering that the responses to whisker deflection, while weak and delayed, were shown to occur, when present, before 50 ms in the Pom (Diamond et al., 1992), it is not clear what the authors mean and consider as "Sensory Responses"? 

We have addressed this important concern in three ways. First, we have reanalyzed our data to include the 50ms pre- and post-stimulus time windows that were previously excluded. This did not qualitatively change our results, but updated statistical measurements are reflected in the Results and the legends of figures 3 and 7. Second, we have created a new figure (new Figure 4) which provides a more detailed analysis of early POm stimulus responses at a finer time scale. Third, we have amended the language throughout the paper to refer to “stimulus responses” rather than “sensory responses” to reflect how we cannot disambiguate between bottom-up sensory input and top-down input into POm and LP with our experimental setup. We refer only to “putative sensory responses” when discussing lowlatency (<100ms) stimulus responses.

Precise wording may help to clarify the message. For instance, line #134: "Of cells from tactilely conditioned mice, 175 (50.4%) significantly responded to the air puff, as defined by having a firing rate significantly different from baseline within one second from air puff onset (Figure 3d, bottom)", could be written "significantly responded to the air puff" should be written "significantly increased (or modified if some decreased) their firing rate within one second after the air puff onset (baseline: ...)". This will avoid any confusion with the sensory responses per se.

We have made this specific change suggested by the reviewer (lines 145-146) and made similar adjustments to the language throughout the manuscript to better communicate our analysis methods. 

(2) To extend the previous concern, the latency of the modulation of the firing rate of the Pom cells for each modality and each conditioning may be an issue. This latency, given in Figure S2, is rather long, i.e. particularly late latencies for the whisker system, which is completely in favor of non-sensory "responses" per se and the authors' hypothesis that sensory-, arousal-, and movement-evoked activity in Pom are shaped by associative learning. Latency is a key point in this study. 

Therefore, 

- latencies should be given in the main text, and Figure S2 could be considered for a main figure, at least panels c, d, and e, could be part of Figure 3. 

- the Figure S2b points out rather short latency responses to the air puff, at least in some cells, in addition to late ones. The manuscript would highly benefit from an analysis of both early and late latency components of the "responses" to air puffs and drafting grating in both conditions. This analysis may definitely help to clarify the authors' message. Since the authors performed unit recordings, these data are accessible.

- it would be highly instructive to examine the latency of the modulation of Pom cells firing rate in parallel with the onset of each behavior, i.e. modification of pupil radius, whisking amplitude, lick rate (Figures 1e, g and 3a, b). The Figure 1 does not provide the latency of the licks in conditioned mice.

- the authors mention in the discussion low-latency responses, e.g., line #299: "In both tactilely and visually conditioned mice, movement could not explain the increased firing rate at air puff onset. These low-latency responses across conditioning groups is likely due in part to "true" sensory responses driven by S1 and SpVi."; line #306: "Like POm, LP displayed varied stimulus-evoked activity that was heavily dependent on conditioning. LP responded to the air puff robustly and with low latency, despite lacking direct somatosensory inputs."  But which low-latency responses do the authors refer to? Again, this points out that a robust analysis of these latencies is missing in the manuscript but would be helpful to conclude.

We have moved our analysis of stimulus response latency in POm to new Figure 4 in the main text and have expanded both the Results and Discussion sections accordingly. We have also analyzed the lick latency on the day of recording, included in a new supplemental Figure S1. 

(3) Anatomical locations of recordings in the dorsal part of the thalamus. Line #122 "Our recordings covered most of the volume of POm but were clustered primarily in the anterior and medial portions of LP (Figure 2d-f). Cells that were within 50 µm of a region border were excluded from analysis." 

How did the authors distinguish the anterior boundary of the LP with the LD nucleus just more anterior to the LP, another higher-order nucleus, where whisker-responsive cells have been isolated (Bezdudnaya and Keller, 2008)? 

Cells within 50µm of any region boundary were excluded, including those at the border of LP and LD. We also reviewed our histology images by eye and believe that our recordings were all made posterior of LD. 

(4) The mention in the Methods about the approval by an ethics committee is missing.  All the surgery (line #381), i.e., for the implant, the craniotomy, as well as the perfusion, are performed under isoflurane. But isoflurane induces narcosis only and not proper anesthesia. The mention of the use of analgesia is missing. 

We thank Reviewer 2 for drawing our attention to this oversight. All experiments were conducted under the approval of the Columbia University IACUC. Mice were treated with the global analgesics buprenorphine and carprofen, the local analgesic bupivacaine, and anesthetized with isoflurane during all surgical procedures. We have amended the Methods section to include this information (Lines 458-470).

Reviewer #3 (Public Review): 

Petty and Bruno ask whether activity in secondary thalamic nuclei depends on the behavioral relevance of stimulus modality. They recorded from POm and LP, but the weight of the paper is skewed toward POm. They use two cohorts of mice (N=11 and 12), recorded in both nuclei using multi-electrode arrays, while being trained to lick to either a tactile stimulus (air puff against whiskers, first cohort) or a visual stimulus (drifting grating, second cohort), and ignore the respective other. They find that both nuclei, while primarily responsive to their 'home' modality, are more responsive to the relevant modality (i.e. the modality predicting reward). 

Strengths: 

The paper asks an important question, it is timely and is very well executed. The behavioral method using a delayed lick index (excluding impulsive responses) is well worked out. Electrophysiology methods are state-of-the-art with information about spike quality in Figure S1. The main result is novel and important, convincingly conveying the point that encoding of secondary thalamic nuclei is flexible and clearly includes aspects of the behavioral relevance of a stimulus. The paper explores the mapping of responses within POm, pointing to a complex functional structure, something that has been reported/suggested in earlier studies. 

Weaknesses: 

Coding: It does not become clear to which aspect of the task POm/LP is responding. There is a motor-related response (whisking, licking, pupil), which, however, after regressing it out leaves a remaining response that the authors speculate could be sensory.

Learning: The paper talks a lot about 'learning', although it is only indirectly addressed. The authors use two differently (over-)trained mice cohorts rather than studying e.g. a rule switch in one and the same mouse, which would allow us to directly assess whether it is the same neurons that undergo rule-dependent encoding. 

We disagree that our animals are “overtrained,” as every mouse was fully trained within 13 days. We agree that it would be interesting to study a rule-switch type experiment, but such an experiment is not necessary to reveal the profound effect that conditioning has on stimulus responses in POm and LP. 

Mapping: The authors treat and interpret the two nuclei very much in the same vein, although there are clear differences. I would think these differences are mentioned in passing but could be discussed in more depth. Mapping using responses on electrode tracks is done in POm but not LP.

The mapping of LP responses by anatomical location is presented in the supplemental Figure S4 (previously S3). We have expanded our discussion of LP and how it might differ from POm.

Reviewer #1 (Recommendations For The Authors):  

Minor writing issues: 

122 ...67 >LP< cells?

301 plural "are”

We have fixed these typos.

Figure issues

*  3a,b time ticks are misaligned and the grey bar (bottom) seems not to align with the visual/tactile stimulus shadings.

*  legend to Figure 3b refers to Figure 1c which is a scheme, but if 1g is meant, this mouse does not seem to have a session 12? 

*  3c,e time ticks slightly misaligned. 

*  5e misses shading for the relevant box plots, assuming it should be like Figure 3h.  

We thank Reviewer 1 for pointing out these errors. We have adjusted Figures 1, 3, and 5 accordingly.

Analyses 

I am missing a similar summary statistics for LP as in Figure 3h 

We have added a summary box chart of LP stimulus responses (Figure 7g), similar to that of POm in Figure 3. We have also performed similar statistical analyses, the results of which are presented in the legend for Figure 7. 

Reviewer #2 (Recommendations For The Authors): 

More precisions are required for the following points: 

(1) The mention of the use of analgesia is missing and this is not a minor concern. Even if the recordings are performed 24 hours after the surgery for the craniotomy and screw insertion and several days after the main surgery for the implant, taking into account the pain of the animals during surgeries is crucial first for ethical reasons, and second because it may affect the data, especially in Pom cells: pain during surgery may induce the development of allodynia and/or hyperalgesia phenomenae and Pom responses to sensory stimuli were shown to be more robust in behavioral hyperalgesia (Masri et al., 2009).  

We neglected to include details on the analgesics used during surgery and post-operation recovery in our original manuscript. Mice were administered buprenorphine, carprofen, and bupivacaine immediately prior to the head plate surgery and were treated with additional carprofen during recovery. Mice were similarly treated with analgesics for the craniotomy procedure. Mice were carefully observed after craniotomy, and we saw no evidence of pain or discomfort. Furthermore, mice performed the behavior at the same level pre- and postcraniotomy (now presented in Figure 1j), which also indicates that they were not in any pain. 

(2) The head-fixed preparation is only poorly described.

Line #414: "Prior to conditioning, mice were habituated to head fixation and given ad libitum water in the behavior apparatus for 15-25 minutes." 

And line #425 "Mice were trained for one session per day, with each session consisting of an equal number of visual stimuli and air puffs. Sessions ranged from 20-60 minutes and about 40-120 of each stimulus. " 

More details should be given about the head-fixation training protocol. Are 15-25 minutes the session time duration, 60 minutes, or other time duration? How long does it take to get mice well trained to the head fixation, and on which criteria?  

Line #389: "Mice were then allowed to recover for 24 hours, after which the sealant was removed and recordings were performed. At the end of experiments,"

The timeline is not clear: is there one day or several days of recordings? 

We have expanded on our description of the head fixation protocol in the Methods. We describe in more detail how mice were habituated to head fixation, the timing of water restriction, and the start of conditioning/training (Habituation and Conditioning, lines 492-500).

(4) Line #411: "Mice were deprived of water 3 days prior to the start of conditioning" followed by line #414 "Prior to conditioning, mice were habituated to head fixation and given ad libitum water in the behavior apparatus for 15-25 minutes".

If I understood correctly, the mice were then not fully water-deprived for 3 days since they received water while head-fixed. This point may be clarified. 

We addressed these concerns in the changes to the Methods section mentioned in the preceding point (3).

(5) Line #157: "Modality selectivity varies with anatomical location in Pom" while the end of the previous paragraph is "This suggests that POm encoding of reward and/or licking is insensitive to task type, an observation we examine further below."

The authors then come to anatomical concerns before coming back to what the Pom may encode in the following section. This makes the story quite confusing and hard to follow even though pretty interesting.  

We have reordered our Figures and Results to improve the flow of the paper and remove this point of confusion. We now present results on the encoding of movement before analyzing the relationship between POm stimulus responses and anatomical location. What was old Figure 5 now precedes what was old Figure 4.

(6) Licks Analysis. Line #99 "However, this mouse also learned that the air puff predicted a lack of reward in the shaping task, as evidenced by withholding licking upon the onset of the air puff. The mouse thus displayed a positive visual lick index and a negative tactile lick index, suggesting that it attended to both the tactile and visual stimuli (Figure 1f, middle arrow)."

Line #105 "All visually conditioned mice exhibited a similar learning trajectory (Figure 1i left, 1j left)". 

Interestingly, the authors revealed that mice withheld licking upon the onset of the air puff in the visual conditioning, which they did not do at the onset of the drifting grating in the tactile conditioning. This withholding was extinguished after the 8th session, which the authors interpret as the mice finally ignoring the air puff. Is this effect significant, is there a significant withholding licking upon the onset of the air puff on the 12 tested mice? 

The withholding of licking was significant (assessed with a sign-rank test) in visually conditioned mice prior to switching to the full version of the task. Indeed, it was the abolishment of this effect after conditioning with the full version of the task that was our criterion for when a mouse was fully trained. We have elaborated on this in the Habituation and Conditioning section in the Methods.

(1) Throughout the manuscript "Touch" is used instead of passive whisker deflection, and may be confusing with "active touch" for the whisker community readers. I recommend avoiding using "touch" instead of "passive whisker deflection".

We appreciate that “touch” can be an ambiguous term in some contexts. However, we have limited our use of the word to refer to the percept of whisker deflection; we do not describe the air puff stimulus as a “touch.” We respectfully would like to retain the use of the word, as it is useful for comparing somatosensory stimuli to visual stimuli.

(2) Line #395: "Air puffs (0.5-1 PSI) were delivered through a nozzle (cut p1000 pipet tip, approximately 3.5mm diameter aperture)".

Are air puffs of <1 PSI applied, not <1 bar?  

We thank Reviewer 3 for pointing out this inaccuracy. The air puffs were indeed between 0.5 and 1 bar, not PSI. We have addressed this in the Methods.

(3) Line #441: "In the full task, the stimuli and reward were identical, but stimuli were presented at uncorrelated and less predictable intervals."  Do the authors mean that all stimuli are rewarded?  

The stimuli and reward were identical between the shaping and full versions of the task. In the full version of the task, the unrewarded stimulus was truly uncorrelated with reward, rather than anticorrelated. 

(4) Line #445 "for a mean ISI of 20 msec." ISI is not defined, I guess that it means interstimulus interval. Even if pretty obvious, to avoid any confusion for future readers, I would recommend using another acronym, especially in a manuscript about electrophysiology, since ISI is a dedicated acronym for inter-spike interval. 

We have defined the acronym ISI as “inter-stimulus interval” when first introduced in the results (Line 82) and in the Methods (Line 511).

(5) Line #416 "In the first phase of conditioning ("shaping"), mice were separated into two cohorts: a "tactile" cohort and a "visual" cohort. Mice were presented with tactile stimuli (a two-second air puff delivered to the distal whisker field) and visual stimuli (vertical drifting grating on a monitor). Throughout conditioning, mice were monitored via webcam to ensure that the air puff only contacted the whiskers and did not disturb the facial fur nor cause the mouse to blink, flinch, or otherwise react - ensuring the stimulus was innocuous. The stimulus types were randomly ordered. In the visual conditioning cohort, the visual stimulus was paired with a water reward (8-16µL) delivered at the time of stimulus offset. In the tactile conditioning cohort, the reward was instead paired with the offset of the air puff. Regardless of the type of conditioning, stimulus type was a balanced 50:50 with an inter-stimulus interval of 8-12 seconds (uniform distribution)." 

The mention of the "full version of the task" will be welcome in this paragraph to clarify what the task is for the mouse in the Methods part.

We have more clearly defined the full version of the task in a later paragraph (line 506). We believe this addresses the potential confusion caused by the original description of the conditioning paradigm. 

(6) Line #467: "Units were assigned to the array channel on which its mean waveform was largest". 

Should it read mean waveform "amplitude"? 

This is correct, we have adjusted the statement accordingly. 

(7) Line #482 "The eye camera was positioned on the right side of the face and recorded at 60 fps." Then line #487 "The trace of pupil radius over time was smoothed over 5 frames (8.3 msec).” 5 frames, with a 60fps, represent then 83 ms and not 8.3 ms.

We have corrected this error.  

(8) Line #121: "257 POm cells and 67 cells from 12 visually conditioned mice" 

67 LP cells, LP is missing 

We have corrected this error. 

(9) Line #354: "A consistent result of attention studies in humans and nonhuman primates is the enhancement of cortical and thalamic sensory responses to an attended visual stimuli. Here, we show not just enhancement of sensory responses to stimuli within a single modality, but also across modalities. It is worth investigating further how secondary thalamus and high-order sensory cortex encode attention to stimuli outside of their respective modalities. Our surprising conclusion that the nuclei are equivalently activated by behaviorally relevant stimuli is nevertheless compatible with these previous studies."  Since higher-order thalamic nuclei are integrative centers of many cortical and subcortical inputs, they cannot be viewed simply as relay nuclei, and there is therefore no "surprising" conclusion in these results. Not surprising, but still an elegant demonstration of the contextdependent activity/responses of the Pom/LP cells. 

We disagree. Visual stimuli activating strong POm responses and tactile stimuli activating strong LP responses - however they do it - is a surprising result. We agree that higher-order thalamic nuclei are integrative centers, but exactly what they integrate and what the integrated output means is still poorly understood.

would this count

This happens in De Quincey's narrative as Betty is warned by the bishop

This is basically the lack of control causing an anxiety of the strange

Traveling causing the surveillance of the government

cjwejcfwejfjwejfdwejfdjwedfwe

The nation attack of fundamental supports of Jim Crow segregation creating the greatest social movement in this nations history.

This helped America recover economically because they gave builders work with these new neighborhoods and gave them better income to pay taxes.

Traditional understanding of what it means to be an American.

The United States gets more involved in atomic bombs and nuclear testing which lead to many health issues and environmental impact.

Marshall's plan must have been a big relief for Americans as it gave us allies if the Soviet Union attacks the US.

How would the nature of warfare be different today if the U.S. hadn't launched the attack at Hiroshima and Nagasaki?

older biblical interpretations did not tie occupation to a means of gaining salvation

rise of the term calling - fulfilment of duty in worldly affairs as the highest form of moral action

capitalism too specific to place under umbrella category of rationalism- must figure out what led to this type of rationalism specifically

not just material conditions or consumption bc same values of capitalism excited in the back woods

older capitalism would not have considered it "moral"- its ethicality ran the risk of clashing with the church

adaptation to capitalist mindset in order to survive is a modern phenom- there was once a time when a capitalist mindset wasn't necessary to survive

America entered war in a unfit state of economic depression. America grew strong but stalin's Soviet unions and the threat of use of nuclear weapons would stop this quickly

In America, this must have been great news as it was one of the biggest issues during the war and used most of our resources.

Nice line

lol

When reactions were performed in solvents such as cyclohexane and cyclooctane, it was found that borylation occurred on the substrate rather than on cyclohexane or cyclooctane. Between cyclohexane and cyclooctane, cyclooctane was more inert towards borylation. Therefore, all reactions were conducted in cyclooctane as the solvent.

When the ligand is 2-methylphenanthroline, the catalyst is way more reactive than when coordinated by other ligands and shows selectivity for primary C-H bonds.

Borylations carried out in the presence of iridium catalysts are selective for primary C-H bonds in a wide range of substrates.

In a glove box under inert atmosphere, catalyst [Ir[Cod][OMe]2], 2-mphen and B2Pin2 were added. A suitable alkane (substrate) and cyclooctanol (solvent) were added and the vial sealed with a Teflon cap. Reaction was heated for 100 degrees Celsius for 20 hours. The reaction was cooled. Product was isolated and purified by column chromatography. Product was analyzed by proton NMR spectroscopy.

In the 3B1B video, Grant Sanderson uses this language in the opposite senses (he talk about adjectives, keys, attending to nouns, queries). As far as we can tell, this is incorrect (there was some discussion about it in the comments.

Note that the way the matrices are shown in the video is consistent with is compatible with K^\top Q (rather than Q K^\top)

The idea of lower nature Cross of light

Looking at how the technology has evolved and where it's headed, I always loved looking at assistive technology. Although I did not understand that assistive technology could include things like glasses or stimulants because of how normalized they are compared to other technology like electronic prosthetics.

I've basically gone my entire life not realizing that I had autism until an autistic friend pointed it out to me. Over my life, I've come up with strategies to control my behavior in social situations. On one hand, I don't think of myself as disabled. But on the other hand, knowing that I was autistic did make feel like a huge weight has been taken off. So many of the things I struggled with in middle school now make so much more sense.

It is actually interesting to know that there are different ways of seeing more colors outside of being color blind. I wonder what type of sight I have and whether I'm a tetrochromat or trichromat.

There is definitely a stigma around the word disability, due to how people with disabilities are treated. Many people think of disabilities, not as a result of society not being designed for a person's needs, but as that person being inept in some way. As a result, many shy away from the label. It also doesn't help that many mental disabilities are also used as insults.

While Zippo lighter fluid (Naphtha) is a bit more expensive per ounce than other solvents (mineral spirits, lacquer thinner, et al), it does usually come packaged in a dispensing container that may make it easier to dispense in a directed method into the internals of typewriters for cleaning them out.

The other benefit is that some may have it on hand for general household use without needing to make a separate trip to the hardware store.

---

via, but not really directly suggested by https://old.reddit.com/r/typewriters/comments/1g9ntnj/lubricant_reccomendations/

Wolves are highly adaptable predators, meaning their hunting strategies can shift based on environmental conditions and prey availability. What factors contribute to the opportunistic hunting behavior of wolves, and how does this flexibility impact their predation on livestock? How do the predator-avoidance strategies of wild prey compare to those of cattle, and what implications does this have for their vulnerability to wolves?

eLife Assessment

This paper addresses an important topic (normative trajectory modelling), seeking to provide a method aiming to accurately reflect the individual deviation of longitudinal/temporal change compared to the normal temporal change characterized based on a pre-trained population normative model. The evidence provided for the new methods is, however, incomplete, with the simulations validating the method needing to be extended.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors provide a method aiming to accurately reflect the individual deviation of longitudinal/temporal change compared to the normal temporal change characterized based on pre-trained population normative model (i.e., a Bayesian linear regression normative model), which was built based on cross-sectional data. This manuscript aims at solving a recently identified problem of using normative models based on cross-sectional data to make inferences about longitudinal change.

Strengths:

The efforts of this work make a good contribution to addressing an important question of normative modeling. With the greater availability of cross-sectional studies for normative modeling than longitudinal studies, and the inappropriateness of making inferences about longitudinal subject-specific changes using these cross-sectional data-based normative models, it's meaningful to try to address this gap from the aspect of methodological development.

In the 1st revision, the authors added a simulation study to show how the performance of the classification based on z-diff scores relatively changes with different disruptions (and autocorrelation). Unfortunately, in my view this is insufficient as it only shows how the performance of using z-diff score relatively changes in different scenarios. I would suggest adding the comparison of performance to using the naïve difference in two simple z-scores to first show its better performance, which should also further highlight the inappropriate use of simple z-scores in inferring within-subject longitudinal changes. Additionally, Figure 1 is hard to read and obtain the actual values of the performance measure. I would suggest reducing it to several 2-dimensional figures. For example, for several fixed values of rho, how the performance changes with different values of the true disruption (and also adding the comparison to the naïve method (difference in two z-scores)).

I would also suggest changing the title to reflect that the evaluation of "intra-subject" longitudinal change is the method's focus.

Author response:

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

Reviewer #1 (Public Review):

The models described are not fundamentally novel, essentially a random intercept model (with a warping function), and some flexible covariate effects using splines (i.e., additive models).

We respectfully but strongly disagree with the reviewer’s assessment of the novelty of our work. The models referred to by the reviewer as “random intercept models … and some flexible covariate effects” seem to relate to the estimation of normative models derived cross-sectionally as developed in and adopted from previous work, not to the work presented here. To be clear, the contributions of this work are: (i) a principled methodology to make statistical predictions for individual subjects in longitudinal studies based on a novel z-diff score, (ii) an approach to transfer information large scale normative models estimated on large scale cross-sectional data to longitudinal studies (iii) an extensive theoretical analysis of the properties of this approach and (iv) empirical evaluation on an unpublished psychosis dataset. Put simply, we provide the ability to estimate within subject change in normative models which until now only provide the ability to show a subject's position in the normative range at a given timepoint. With the exception of the reference [13] cited in the main text, we are not aware of any methods available that can achieve this. Based on this feedback combined with the feedback of the Reviewer 2, we now improved our introduction and clearly state our contribution right from the outset of the manuscript whilst also shortening the introduction to make it more concise. In this work, we are trying to be very transparent in showing to the reader that our method builds on a previously peer-reviewed model.

The assumption of constant quantiles is very strong, and limits the utility of the model to very short term data.

We now provide an extensive theoretical analysis of our approach (section 2.1.3), where we show that this assumption is actually not strictly necessary and that our approach yields valid inferences even under much milder assumptions. More specifically, we first provide a mathematical grounding for the assumption we made in the initial submission, then generalise our method to a wider class of residual processes and show that our original assumption of constant quantiles is not too restrictive. We also provide a simulation study to show how the practitioner can evaluate the validity and implications of this assumption on a case-by-case basis. This generalisation is described in depth in section 2.1.3.

The schizophrenia example leads to a counter-intuitive normalization of trajectories, which leads to suspicions that this is driven by some artifact of the data modeling/imaging pipelines.

We understand that the observed normalisation effects might appear surprising. As we outlined in our provisional response, we would like to emphasise that there is increasing evidence that the old neurodegenerative view of psychosis is an oversimplification and that trajectories of cortical thickness are highly variable across different individuals after the first psychotic episode. More specifically, we have shown in an independent sample and with different methodology that individuals treated with second-generation antipsychotics and with careful clinical follow-up can show normalisation of cortical thickness atypicalities after the first episode (https://www.medrxiv.org/content/10.1101/2024.04.19.24306008v2, now accepted in Schizophrenia Bulletin). These results are well-aligned with the results we show in this manuscript. We now added remarks on this topic into the discussion. We would also like to re-emphasise that the data were processed with the utmost rigour using state of the art processing pipelines including quality control, which we have reported as transparently as possible. The confidence that the results are not ‘driven by some artifact of the data modeling/imaging pipelines’ is also supported by the fact that analysis of a group of healthy controls did not show any significant z-diffs (see Discussion section), neither frontally nor elsewhere. If the reviewer believes there are additional quality control checks that would further increase confidence in our findings, we would welcome the reviewer to provide specific details.

The method also assumes that the cross-sectional data is from a "healthy population" without describing what this population is (there is certainly every chance of ascertainment bias in large scale studies as well as small scale studies). This issue is completely elided over in the manuscript.

Indeed, we do not describe the cross-sectional population used for training the models, as these models were already trained and published with in-depth description of the datasets used for the training (https://elifesciences.org/articles/72904). We now make this more explicit in the section 2.1.1. of the manuscript (page 7), and also more explicitly acknowledge the possibility of ascertainment bias in the simulation section 2.1.4. However, we would like to emphasise that such ascertainment bias is not in any way specific to the analyses we report. In fact it is present in all studies that utilise large scale cohorts such as UK Biobank. Indeed, we are currently working on another manuscript to address this question in detail, but given the complexity of this problem and the fact that many publicly available legacy studies simply do not record sufficient demographic information, e.g. to assess racial bias properly, we believe that this is beyond the scope of the current work.

Reviewer #2 (Public Review):

The organization and clarity of this manuscript need enhancement for better comprehension and flow. For example, in the first few paragraphs of the introduction, the wording is quite vague. A lot of information was scattered and repeated in the latter part of the introduction, and the actual challenges/motivation of this work were not introduced until the 5th paragraph.

As noted above in our response to Reviewer 1, we significantly pruned the introduction, stating our objective in the first paragraph and elaborating on the topic later in the text. We hope that it is now less repetitive and easier to follow.

There are no simulation studies to evaluate whether the adjustment of the crosssectional normative model to longitudinal data can make accurate estimations and inferences regarding the longitudinal changes. Also, there are some assumptions involved in the modeling procedure, for example, the deviation of a healthy control from the population over time is purely caused by noise and constant variability of error/noise across x_n, and these seem to be quite strong assumptions. The presentation of this work's method development would be strengthened if the authors can conduct a formal simulation study to evaluate the method's performance when such assumptions are violated, and, ideally, propose some methods to check these assumptions before performing the analyses.

This comment encouraged us to zoom out from our original assumption and generalise our method to a wider class of residual processes (stationary Gaussian processes) in section 2.1.3. We now present a theoretical analysis of our model to show that our original assumption (of stable quantiles plus noise) is actually not necessary for valid inference in our method, which broadens the applicability of our method. Of course, we also discuss in what way the original assumption is restrictive and how it aligns with the more general dynamics. We also include a simulation study to evaluate the method's performance and elucidate the role of the more general dynamics in section 2.1.4.

The proposed "z-diff score" still falls in the common form of z-score to describe the individual deviation from the population/reference level, but now is just specifically used to quantify the deviation of individual temporal change from the population level. The authors need to further highlight the difference between the "z-score" and "z-diff score", ideally at its first mention, in case readers get confused (I was confused at first until I reached the latter part of the manuscript). The z-score can also be called a measure of "standardized difference" which kind of collides with what "z-diff" implies by its name.

We added the mention of the difference between z-score and z-diff score into the last paragraph of introduction.

Explaining that one component of the variance is related to the estimation of the model and the other is due to prediction would be helpful for non-statistical readers.

We now added an interpretation of the z-score in the original model below equation 7.

It would be easier for the non-statistical reader if the authors consistently used precision or variance for all variance parameters. Probably variance would be more accessible.

This was a very useful observation, we unified the notation and now only use variance.

The functions psi were never explicitly described. This would be helpful to have in the supplement with a reference to that in the paper.

Indeed, while describing the original model we had to make choices about how to condense the necessary information from the original model so that we can build upon it. As the phi function is only used for data transformation in the original model, we did not further elaborate on it, however, we now refer to the specific section of the original paper of Fraza et al. 2021 where it is described more in detail (https://www.sciencedirect.com/science/article/pii/S1053811921009873).

What is the goal of equations (13) and (14)? The authors should clarify what the point of writing these equations is prior to showing the math. It seems like it is to obtain an estimate of \sigma_{\ksi}^2, which the reader only learns at the end.

We corrected the formatting.

What is the definition of "adaption" as used to describe equation (15)? In this equation, I think norm on subsample was not defined.

We added a more detailed description of the adaptation after equation 15.

"(the sandwich part with A)" - maybe call this an inner product so that it is not confused with a sandwich variance estimator. This is a bit unclear. Equation (8) does have the inner product involving A and \beta^{-1} does include variability of \eta. It seems like you mean that equation (8) incorrectly includes variability of \eta and does not have the right term vector component of the inner product involving A, but this needs clarifying.

We now changed the formulation to be less confusing and also explicitly clarified the caveat regarding the difference of z-scores.

One challenge with the z-diff score is that it does not account for whether a person sits above or below zero at the first time point. It might make it difficult to interpret the results, as the results for a particular pathology could change depending on what stage of the lifespan a person is in. I am not sure how the authors would address those challenges.

We agree with the outlined limitation in interpretation of overall trends when the position in the visit one is different between the subjects. However, this is a much broader challenge and is not specific to our approach. This effect is generally independent of the lifespan, but may further interact with the typical lifespan of disease. rWhen the z scores are taken in the context of the cross-sectional normative models, it does make it possible to identify what the overall trend of an illness is across the lifespan, and individual patient’s z-diffs not in line (with what would this typical group trajectory predicts) may e.g. correspond to early/late onset of their individual atrophy. We now make these considerations explicitly in the discussion section.

Reviewer #2 (Recommendations For The Authors):

Other minor suggestions to help improve the text:...

We thank Reviewer #2 for the list of minor suggestions to improve the text, which we all implemented in the manuscript.

The hardest part of typewriter repair is believing you can do it. Everything else is just instructions plus a careful, thoughtful hand.<br /> —Theodore Munk

There are clear signals that he is starting to lose hope

Where are the voices of the women in the war?

Volle: The firing of a gun or weapon at the same time.

the soldiers were so desperate that they resorted to stealing soap.

Gibson Clough grew up being educated and he decided to enlist on his own.

Expression of skepticism about the inevitability of a societal bifurcation due to AI.

Expression of optimism about the potential benefits of AI.

Discussion on the capabilities of robots to communicate and interact with humans.

Highlighting software as the major challenge in robotics.

Introduction to the three major categories of challenges in robotics.

Discussion on the theory and potential of humanoid robots.

Personal anecdote about owning Chinese robot dogs, illustrating the advancements in robotics.

Discussion on the advancements and current state of self-driving cars.

Highlighting robotics as a major area of interest and inquiry.

Inquiry about the investment strategy and industries most advantaged by AI.

Comparison of AI's impact to other major technological advancements like the microprocessor, computer, and internet.

Discussion on the importance of rapid technological progress from a political standpoint.

Introduction of the guest, Mark Andre, highlighting his role as an innovator.

Introduction to the topic of AI censorship, emphasizing its future intensity and importance.

It's interesting that the same words are inherently viewed as more formal when written down vs. spoken by all three class divisions.

This reminds me of the Columbian exchange and how with the addition of new plants and animals brought from the Old World, it also came with diseases and new definitions of unsanitary. This was about 100 years after Columbus' first expedition, so this isn't a novel idea about taking the old into the new. Even if he's not specifically talking about plants and is referring to social roots as opposed to physical materials, his statement is nonetheless true. The cultures colliding, already knowing that people inhabit the land, will be a point of contention for the British as they continue to explore America. The British would be spreading their beliefs and culture across the land while the Indigenous population would have to learn to assimilate (or vehemently oppose) to this new way of living.

Who this text is for: the Queen and her advisors. It's been well established that the purpose of this document was to list the ways that England would benefit from expanding into the colonies, but there's no way to know what could have come from such making such an expedition. the Queen may have been hesitant to spend more money and resources for this voyage and wanted to focus on her subjects in the mother country before advancing to a new land. If she had any doubts before, I would say they went away pretty quickly after Hakluyt's reasoning for wanting England to colonize new lands.

Something to take note of when reading is that there is a very specific audience in mind when Hakluyt was writing this. This was written to persuade someone, her Majesty Queen Elizabeth I. There is no taking into account how this will affect the people already inhabiting the land, what colonizing the land would even look like. It's a letter written with the sole intention of convincing her to spend more resources and supplies to secure creating a settlement that will procure wealth and religious prosperity for the Queen, but also Great Britain as a whole. Native Americans are not a part of that equation.

Did the Spanish have a different religion than that of the Englishmen? This tenant implies that the English would have to give up their religion when interacting with the Spanish. By doing so, they're also abandoning their loyalty to the Queen. Would they be punished for believing in a different religion than the Spanish? Is this just a hypothetical?

It will be a short voyage and the route they choose to take should not interfere with any trade routes or be near any territory claimed by other Princes.

Very interesting that Hakluyt emphasizes how creating colonies in the Americas will bring in greater revenue for the Queen by creating taxes for the colonists to have to pay. This notion, used more as a persuasion technique than anything concrete, is the catalyst for the American Revolution that occurs almost 200 years later. It's stated outright this is for the Queen's benefit and not the people who will reside there. The implications of that are quite clear, that Britain sees the colonies as a means to an end for their imperialistic endeavors. Wonder what Thomas Paine would think if he saw that...

happening near or around an equinox

One of the huge driving forces behind England's colonization was them wanting to be better than the Spaniards for how they decided to colonize. The Spanish were known for their brutality and unspeakable offenses to the native people of the land, and so many other countries that wished to expand their own territory were adamant on not behaving like them. However they chose to interpret that "we didn't kill or rape as many of them as the Spaniards did", if it was justification as to why they should expand (to spite the Spaniards), or for another reason altogether, I'm not certain. I do know that the Spanish did influence how other countries went about exploring and invading land and served as a "what not to-do" when encountering the people native to the land.

This document was written 92 years after Columbus had made his first voyage to the Americas. English colonization wasn't a brand new idea at this time, so I wonder why Hakluyt had to convince the Queen to devote more of their resources to it (did their voyages not bear fruit?).

1 - The legal size reported for harvest is 160mm in width and the sex of these harvests are male as of 2012. As of 2024, ODFW regulations show that harvests must be only a minimum of 146mm and still must only be male. Harvesters must measure this width using calipers. There is no difference for recreational or commercial dungeons crab harvesting.

For next edition, rewrite all JAGS code so that the order/format matches how we write down equations describing our models. E.g.:

response variable ~ statistical distribution(parameters) transformation(parameters) <- linear predictor

Zeker, hier is de toelichting:

Zelfmisleiding kan nuttig zijn om de voortgang in het herstelproces en de kans op herstel gedurende de behandeling te volgen. Het geeft inzicht in hoe iemand met een verslaving zichzelf en zijn situatie ziet, en kan helpen bepalen hoe realistisch of optimistisch ze over hun eigen herstel zijn.

Impression Management (IM), ofwel de bewuste neiging om zichzelf in een positiever daglicht te stellen, is een misleidend gedrag waarmee rekening moet worden gehouden. IM kan ervoor zorgen dat verslaafden sociaal wenselijke antwoorden geven, bijvoorbeeld door hun problemen te bagatelliseren. Het is belangrijk dit te herkennen en in te calculeren bij het evalueren van hun herstel.

Emotionele manipulatie (EM) moet ook in overweging worden genomen om de aard van de misleidende dynamieken bij verslaving beter te begrijpen. EM geeft inzicht in hoe verslaafden anderen emotioneel proberen te beïnvloeden om hun situatie of gedrag te rechtvaardigen. Dit helpt zorgverleners de complexe sociale en emotionele strategieën te begrijpen die sommige verslaafden gebruiken in hun interacties en herstel.

Zeker! Hier is de toelichting:

Zelfmisleiding heeft invloed op hoe mensen hun mentale gezondheid ervaren, afhankelijk van hun niveau van emotionele manipulatie (EM). Bij mensen met hoge EM en zelfmisleiding zien we twee soorten misleidende patronen:

1. Zichzelf als slachtoffer presenteren: Deze mensen benadrukken vooral de emotionele en mentale negatieve gevolgen van hun verslaving, waardoor ze anderen (en misschien zichzelf) laten zien dat ze lijden onder hun verslaving. Dit kan een manier zijn om sympathie of hulp te krijgen zonder daadwerkelijk stappen te ondernemen om hun situatie te verbeteren.

2. Het overdrijven van hun mentale welzijn: In plaats van de negatieve aspecten te benadrukken, kunnen ze hun geestelijke gezondheid juist te positief inschatten om hun zelfbeeld te beschermen. Ze doen dit om hun zelfvertrouwen op te bouwen en zich beter aan te passen, ook al is hun situatie in werkelijkheid niet zo positief.

Bij mensen met hoge zelfmisleiding en EM kan dit leiden tot passiviteit in hun herstel. Ze kunnen ervoor kiezen om zelf geen oplossingen te zoeken en wachten tot de hulp van buitenaf komt. Dit heeft negatieve gevolgen voor hun behandelsucces en vergroot het risico op terugval, vooral bij degenen met een hoog niveau van emotionele manipulatie.

Bij mensen met een verslaving die geen andere drugs of middelen dan hun hoofddrug noemen, lijkt zelfmisleiding een grotere rol te spelen. Zelfmisleiding houdt in dat ze zichzelf wijsmaken dat hun verslaving minder erg is dan het daadwerkelijk is. Ze doen dit onbewust, omdat het idee van de schade die hun verslaving veroorzaakt, niet past bij hun positieve zelfbeeld. Door zichzelf in bescherming te nemen met deze zelfmisleiding, vermijden ze het onder ogen zien van de problemen en gevaren van hun verslaving.

Dit kan bijvoorbeeld betekenen dat iemand die alleen zijn hoofdmiddel (zoals alcohol) noemt, zichzelf overtuigt dat het allemaal "niet zo erg is" en dat het "niet echt een probleem" is. Ze vertellen zichzelf dat ze het onder controle hebben, ook al heeft de verslaving wel degelijk negatieve gevolgen voor hun leven en gezondheid.

Zeker! Ik zal punten 1 en 3 nog eenvoudiger uitleggen:

Punt 1: Zelfmisleiding en het ontkennen van problemen

Bij mensen met een verslaving die geen andere drugs of middelen dan hun hoofddrug noemen, lijkt zelfmisleiding een grotere rol te spelen. Zelfmisleiding houdt in dat ze zichzelf wijsmaken dat hun verslaving minder erg is dan het daadwerkelijk is. Ze doen dit onbewust, omdat het idee van de schade die hun verslaving veroorzaakt, niet past bij hun positieve zelfbeeld. Door zichzelf in bescherming te nemen met deze zelfmisleiding, vermijden ze het onder ogen zien van de problemen en gevaren van hun verslaving.

Dit kan bijvoorbeeld betekenen dat iemand die alleen zijn hoofdmiddel (zoals alcohol) noemt, zichzelf overtuigt dat het allemaal "niet zo erg is" en dat het "niet echt een probleem" is. Ze vertellen zichzelf dat ze het onder controle hebben, ook al heeft de verslaving wel degelijk negatieve gevolgen voor hun leven en gezondheid.

Punt 3: Emotionele manipulatie bij jonge gebruikers

Bij mensen die al op jonge leeftijd (voor hun 18e) met drugs begonnen, komt vaak emotionele manipulatie (EM) voor. Dit betekent dat ze bewust emoties gebruiken om anderen te beïnvloeden. Ze doen bijvoorbeeld alsof ze zich slechter voelen dan ze eigenlijk doen, of ze spelen slachtoffer om medelijden te wekken. Dit kan een manier zijn om verantwoordelijkheid te vermijden of om anderen zo ver te krijgen om hen te helpen of hun gedrag goed te keuren.

Vaak hangt dit gedrag samen met antisociale kenmerken, zoals de neiging om zich niet veel aan te trekken van regels of van wat anderen vinden. Ze proberen dus met manipulatieve middelen hun zin te krijgen, in plaats van hun problemen openlijk en eerlijk te delen. Dit kan ook te maken hebben met hun verslaving, omdat ze geleerd hebben emoties in te zetten om anderen te overtuigen dat ze hulp of speciale behandeling nodig hebben.

Mensen die geloven in het false memory-perspectief zeggen dat herinneringen aan misbruik op dezelfde manier werken als andere emotionele herinneringen. Als het misbruik veel stress en negatieve emoties veroorzaakte, zouden stresshormonen vrijkomen die ervoor zorgen dat die herinneringen later goed worden onthouden. Bij traumatische ervaringen, zoals bij mensen met PTSS, zien we bijvoorbeeld dat zulke gebeurtenissen vaak goed worden herinnerd.

Volgens dit idee ontstaan valse herinneringen wanneer iemand zegt dat hij zich een traumatische gebeurtenis uit het verleden herinnert, maar eigenlijk echte en ingebeelde gebeurtenissen door elkaar haalt. Dit gebeurt vaak tijdens bepaalde therapieën waarin mensen proberen oude herinneringen op te halen, zoals in zogenaamde "recovered memories therapy".

Vervalsing gebaseerd op medische aandoeningen kan in de meeste gevallen worden gecombineerd met vervalsing in andere domeinen, zoals het psychologische en cognitieve. Dit hoofdstuk benadrukt vooral de noodzaak om, vooral bij complexe medische en somatische klachten, meerdere benaderingen te volgen om simulatie/veinzen correct te beoordelen. Het beoordelen van zorgdossiers, uitgebreide interviews en psychologisch testen vormen de basis voor de systematische evaluatie van responsstijlen.

Het woord "feigning" (simuleren) betekent dat iemand bepaalde kenmerken, handelingen of ziektes nabootst of doet alsof. Dit gedrag wordt vaak gezien door artsen en psychologen, vooral in gevallen waarbij sprake is van lichamelijke of psychische klachten. In de medische context betekent feigning het overdrijven van symptomen.

Aan de andere kant is "malingering" (simulatie) een bewuste keuze om opzettelijk symptomen te overdrijven of een medische of psychologische aandoening te verzinnen, meestal met het doel om er extern voordeel uit te halen, zoals geld van de zorgverzekering, eerder met pensioen gaan, of minder werken.

Hoewel feigning en malingering op elkaar lijken, is feigning een bredere term. Feigning kan plaatsvinden zonder duidelijk bewijs van extern gewin en kan voortkomen uit onwetendheid over het eigen gedrag, terwijl malingering altijd te maken heeft met opzettelijke misleiding om er zelf voordeel uit te halen.

Drie vormen van feigning: Feigning kan op drie manieren voorkomen:

1. Lichamelijk (somatisch): Het overdrijven of verzinnen van lichamelijke klachten of ziektes.
2. Cognitief: Het overdrijven van cognitieve problemen zoals geheugenverlies of concentratieproblemen.
3. Psychologisch (emotioneel): Het nabootsen van psychische klachten, zoals angst of depressie.

Drie vormen van malingering volgens Resnick (1984): 1. Pure malingering: De persoon verzint een psychologische of medische aandoening die nooit heeft bestaan. 2. Partiële malingering: De persoon overdrijft symptomen van een aandoening die wel bestaat. 3. Valse toeschrijving: De persoon wijt de symptomen aan een ongerelateerde oorzaak. In dit geval zijn de symptomen echt, maar de oorzaak wordt verkeerd weergegeven

Een kopie van het rapport verstrekken: Er is geen consensus onder clinici over het al dan niet verstrekken van kopieën van neuropsychologische assessmentrapporten aan patiënten. Hoewel de wet patiënten doorgaans toegang verleent tot hun medische en meeste geestelijke gezondheidsdossiers, mogen neuropsychologen onder de richtlijnen van de Health Insurance Portability and Accountability Act (HIPAA) rapporten achterhouden als het verstrekken ervan aanzienlijke schade aan de patiënt kan veroorzaken.

Klachten afhandelen: Patiënten kunnen de feedback niet waarderen, van streek raken en dreigen een klacht in te dienen bij bijvoorbeeld een ethische commissie. Neuropsychologen wordt aangeraden om een kopie van eventuele klachten op te slaan. Klachten kunnen namelijk onnauwkeurige informatie bevatten of schadelijke taal die in een verdediging kan worden gebruikt. In neuropsychologische settings worden klachten vaak ingediend wanneer patiënten overtuigd zijn dat ze geen extern voordeel hebben en/of verantwoordelijkheid willen vermijden. Vanwege de verschillende aard van klachten kan het medische model voor klachtenbehandeling niet worden gebruikt binnen neuropsychologische evaluaties. Wanneer het bovengenoemde feedbackmodel volledig en correct is toegepast en er toch een klacht wordt ingediend, kan de neuropsycholoog verder niets meer doen.

Alternatieve zienswijzen: Er bestaat geen universele benadering voor het geven van feedback aan patiënten over testprestaties. Neuropsychologen verschillen in hoeveel informatie ze delen over inspanningstests. De kern van de hierboven beschreven procedure is daarom het consequent benadrukken van een patiëntgerichte aanpak.

Zoals vermeld in de handleiding van de Test of Memory Malingering, heeft het gebruik van de term "simulatie" op zichzelf meestal weinig waarde voor patiënten en verwijzende instanties. De klinische zorg kan worden verbeterd door de motivatie van de patiënt voor simulatie te onderzoeken en relevante interventies voor te stellen die mogelijk nuttig zijn, ook al bestaat er geen specifieke "behandeling" voor simulatie.

Hier is een samenvatting van de belangrijkste onderdelen van deze tekst:

1. Eerste fase van behandeling: De focus ligt op het stellen van grenzen, het versterken van copingstrategieën en het behouden van vertrouwen. Technieken zoals het bijhouden van dagboeken, familie foto's bekijken, opnames van sessies herhalen en ontspannings- en mindfulness-technieken toepassen, worden als nuttig beschouwd. Deze fase wordt afgesloten als de moeder (dader) kan erkennen dat ze het kind heeft misbruikt, al is het oppervlakkig.

2. Tweede fase: In deze fase begint de moeder empathie voor het kind te ontwikkelen en haar eigen schuld te erkennen, zonder anderen de schuld te geven. Traumatische herinneringen uit haar eigen jeugd kunnen naar boven komen, wat vaak leidt tot familiebreuk.

3. Laatste fase: Deze fase richt zich op het opbouwen van een nieuwe en gezonde band tussen de moeder en het kind. Empathie, volledige erkenning van het misbruik, en het ontwikkelen van een geschikte gehechtheid staan centraal. Veel dader-moeders maken de volledige behandeling echter niet af, en behandeling kan meer dan vijf jaar duren.

4. Therapie voor andere gezinsleden: Volgens het model van Parnell en Day (1998) moeten gezinsleden tegelijk met de dader worden behandeld. De vader krijgt individuele therapie om zijn emoties over het misbruik te verwerken, en het kind volgt vaak speltherapie om zich aan te passen aan de nieuwe gezinsdynamiek. Over behandeling van broers en zussen is weinig bekend.

5. Wettelijke aspecten: Na het vaststellen van het misbruik houdt kinderbescherming toezicht op het gezin, soms jaren lang. Veel moeders krijgen daarnaast te maken met strafrechtelijke vervolging.

Er is nog veel onbekend over dit syndroom, zoals de prevalentie, het verloop en sterftecijfers. Alleen door professionals goed op te leiden, kunnen slachtoffers beter beschermd worden en nauwkeuriger diagnoses gesteld worden.

Na een PCF-diagnose zijn er drie mogelijke vervolgstappen:

1. De arts stelt voor de diagnose als een alternatieve mogelijkheid te beschouwen en uit te sluiten door middel van intensieve observatie.
2. Onderzoekers plaatsen camera's in de ziekenhuisomgeving van het kind of verkrijgen een huiszoekingsbevel.
3. Een arts beoordeelt de medische dossiers van het slachtoffer. Deze kunnen waardevolle informatie bieden over patronen van misbruik.

Bij het aanpakken van een Munchausen by Proxy (MBP)-zaak is een multidisciplinaire aanpak cruciaal. Zodra MBP wordt vermoed, moet alle relevante informatie zorgvuldig verzameld worden om de diagnose van Pediatric Condition Falsification (PCF) te bevestigen of uit te sluiten. Hierbij horen de huidige en eerdere medische gegevens van het kind en de moeder, gesprekken met mensen die het gezin kennen (zoals scholen en kinderopvang), en informatie van familieleden. Onderzoeken gebeuren vaak in ziekenhuizen, soms met videobewaking. Als misbruik wordt vastgesteld, moeten het kind en broers/zussen direct worden beschermd tegen contact met de moeder. Diagnostiek begint vaak met een kinderarts die bepaalt of de symptomen een medische oorzaak hebben; dit kan voldoende zijn om PCF te diagnosticeren.

Dit betekent dat bepaalde huidproblemen die door een persoon op zichzelf worden veroorzaakt – zoals door krabben, branden of andere vormen van zelfbeschadiging – al lang als een erkende medische kwestie worden gezien. Dit toont aan dat zelfbeschadiging van de huid serieus wordt genomen in de medische wereld en dat er al lang aandacht aan wordt besteed.

This header does not have the same header-style as for the previous week. This is for all headers from week 4 to 14. Please set them to header 1

I think because my background is in TESOL, I have been really hesitant to get students to read their work out loud. When you get TESOL training, it tends to emphasize reading out loud as an exercise in pronunciation practice. But this reading (and our discussion about it) really challenged my thinking, especially because I reflected a bit more on my own process as a writer. I read my own work out loud constantly. And I saw the value of doing this with students I was consulting in the Writing Center when I worked as a tutor. I want to incorporate this a bit more in low-stakes ways (maybe as part of student conferences?) to just see how it goes.

There is a different gap-start value than gap-extend value as default. Therefore, the scoring is affine. The end-gap penalty is set to False per default and therefore end-gaps are not penalized, despite using global alignment.

The CDS is the nucleotide sequence of the translated region only. The mRNA also has a 5'UTR, 3'UTR, and Kozak sequence and might even have several ORFs in it.

In this case, the gene has 2 exons that make up 1 ORF.

The Europeans had thought that the Native Americans were carrying around an abundance of cinnamon, however it was not cinnamon. They then showed the Native Americanas what cinnamon was because they had brought some. From there the Native Americans had some idea where more cinnamon was. This would be used for trading.

The Native American diet had made them in such great shape. They would eat lots of protein, from the animals they killed. They also lived off of various plants. They weren't introduced to sugar yet and so why would they not have a good figure. As I mentioned before, they were basically "working out" all day. Making everything that they owned and going off to hunt for their food, they were very active.

Christopher is making note of the materials that they lack. While also seeing how they used different materials in place of such things, such as iron. I would have to think that he was rather impressed by the creativity and how they made life work with what they had. However, he doesn't want to help, he believes that him and his crew should take them in as prisoners.

Christopher and his crew had been given many gifts by the Native Americans, even though the Native Americans expected nothing in return. This baffles me because it was their own land and the Europeans barely gave back, I man they gave them some gifts, but they were so focused on finding gold. Hungry for money, they didn't care to ruin someone else's world.

Here, I wonder if Christopher was scared of this happening to the Europeans. Did he think that this would happen when he first landed on the island? They had to have done intensive research and maybe were spying on them before they actually landed. I can't imagine them coming all that way just o be eaten alive.

Here Christopher talks about enslaving the Native Americans. The importance of this sentence is that this is where the start of ruining tribes and taking over land begins. When he says "subjugated and made to do what is required of them" he is bluntly saying that these people will be his labor. The Native Americans were never required to do anything for the Europeans, then they show up and capture them and use them.

At this point Chris and his crew had been with them on their island for some time, he was watching them and the way they work more so than he had before it seems. A question came into my mind when reading this, I wonder if the Europeans ever helped the Indigenous people? When something like this would happen (their boats breaking) would they sit back and watch, or would they go to help? He goes onto say that he was trying to take them somewhere, but they had no intentions of going. Was that what was on his mind the whole time? Maybe they didn't know how to help.

This could be coming from the "lens" that I am reading from, but I do not trust Christopher whatsoever. He starts out this journal by saying that they can be friends and that everything is going well and that the Native Americans were appreciated of their gifts. Not too long after that he goes on to say how good of servants they can be. Taking them from a friend level all the way down to being his little peasants. Rude. Then he goes on to say that he can convert them to Christianity without learning their own beliefs. He said it seemed as if they had no religion, so how good of friends were they? Did he even take the time to learn the way they live? He had one mission and it's pretty obvious.

Here you can see that there is a lack of respect for the Native Americans. Chris, even though he just met them, was already calling them poor people. However, to the Native Americans, they didn't feel poor. They had everything they needed at that time and made their own way of life, not needing all of the extra "stuff" that the English had. They were rich in the materials that they needed at that time before new things had been introduced.

When Christopher says that the Native Americans were "well made" he was talking about the muscles and strength they had. Because they made everything themselves, they had to be carrying around large objects and using their strength to build various things.

SITC continuously evaluates the field for emerging data and new FDA approvals. Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC Lymphoma Cancer Immunotherapy Guideline Expert Panel. More information on the SITC Guidelines can be found at sitcancer.org/guidelines

An update to the SITC Lymphoma CPG is currently in preparation to address practice-changing data and new FDA approvals of immune checkpoint inhibitors, CAR T cell therapies, antibody-drug conjugates, monoclonal antibodies, bispecific T cell engagers, immunomodulatory agents, and a cytotoxic recombinant protein. These approvals and practice-changing data will affect the recommendations across a variety of disease states within this guideline.

Update 10-22-2022

Based on the approval of axicabtagene ciloleucel for adult patients with large B-cell lymphoma (LBCL) that is refractory to first-line chemoimmunotherapy or relapses within 12 months of first-line chemoimmunotherapy, the Lymphoma CPG has been updated in the following locations: * Available Agents and Indications for NHL * Expert Panel Recommendations for NHL * Expert Panel Recommendations for NHL

Reference: Axicabtagene ciloleucel (YESCARTA) FDA press release

Based on the approval of tisagenlecleucel for the treatment of adult patients with relapsed or refractory follicular lymphoma following 2 or more lines of systemic therapy, the Lymphoma CPG has been updated in the following location: * Available Agents and Indications for NHL

Reference: Tisagenlecleucel (KYMRIAH) FDA press release

Based on the approval of lisocabtagene maraleucel for the treatment of adult patients with large B-cell lymphoma (LBCL) who have refractory disease to first-line chemoimmunotherapy or relapse within 12 months of first-line chemoimmunotherapy; or refractory disease to first-line chemoimmunotherapy or relapse after first-line chemoimmunotherapy and are not eligible for hematopoietic stem cell transplantation (HSCT) due to comorbidities or age, the Lymphoma CPG has been updated in the following location: * Available Agents and Indications for NHL

Reference: Lisocabtagene maraleucel (BREYANZI) FDA press release

Update 6-3-2021

Based on the approval of loncastuximab tesirine-lpyl for the treatment of adult patients with relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, DLBCL arising from low grade lymphoma, and high-grade B-cell lymphoma, the Lymphoma CPG has been updated in the following location: * Available Agents and Indications for NHL

Reference: Loncastuximab tesirine-lpyl (ZYNLONTA) FDA press release

Update 2-9-2021

The Lymphoma CPG has been updated in the following location: * Immunotherapies in Development for NHL * Expert Panel Recommendations for NHL

@astitva Pls refer to the Google Doc for this section to crosscheck and make any necessary corrections.

Third CTA missing @astitva The text/section below should be added under the 2 CTA boxes. Let’s Bring Your Vision to Life! CTA: Contact Us Today.

The supporting image should display the thumbnail of the Maker Monday cover. @astitva

The CTA button should read: Claim Your Free Activities ➜

The CTA button shoudl read: Subscribe Now ➜

Good example of how everything in a theory that we think is truth relates to the concepts of our paradigm.

Competing views about the relation between observations reality and truth. Popper argues that observations help us distinguish which theories are true or not plus bringing us always closer to a more true scientific theory. Wittgenstein argues this can go both ways. Kuhn argues that these are observations are couched in the language of our paradigm and so everything is relative to that.

Defektin uygun malzeme ile restorasyonu

@Terence, I don’t recall changing the grade range from 1-6 to K-8. Originally, it read: “Browse our easy-to-implement lesson plans that make teaching tech a breeze. Perfect for grades 1-6, with new updates on the horizon!” Just flagging this for reference and cross-checking.

born into a state of exile or marginalization as a black woman in the US

I feel that this means that it is the idea of finding strength and comfort within herself and the gain of power to celebrate.

I take this line as they are proud of the life they created for themselves, especially since they had no model to go by. And wants to celebrate that with others.

She refers to the historical and cultural contexts of oppression and marginalization, showing her resilience in overcoming those barriers

starshine is heaven and clay is earth. she is stuck in between the two. stuck between life and death

Lucille shows how she is a Black woman. I believe she says this line because Black women struggle in the real world and she is trying to emphasize that and how they are looked down upon.

the author built themselves a life without a guide of sorts or an instruction manual to get to where they landed

She has faced so many hardships yet has had the strength to prevail through it all.

I see this line as her having to support herself. She doesn't have someone to celebrate with her so she needs to make the effort to ask. This is representative of black women being among the least protected demographics.

Megan is at the highest of the hip-hop game and she's letting it be known.

Here Megan is referencing her previous music deal with 1501 certified entertainment which was a predatory deal that benefitted the label exponentially more than her. Now she's signed to Warner with a deal that benefits the company and her.

Megan is expressing how important her value is, especially as a female rapper of her caliber. She's letting men know her worth and not lowering her light and confidence.

She is basically saying that she needs to step her game if the haters aren't hating up and become a unique person outside of other rappers in the game.

"itchin for a motherfuckin problem" and "go get tested" is referring to an STI test. Megan is calling the people looking for a problem as dirty, the use of "itchin" is in reference to a common STI.

She is showing how fame comes with hate when you are loved by many. People love to hate on successful women.

she knows that being successful comes with hate

Other women treat men like they're special and the most important thing to them, but Megan treats them like a quick snack and she moves on without getting attached.

Here Megan is cleverly referencing a famous colloquialism "everything's bigger in Texas", asserting that her body is all natural (no injections/plastic surgery) and the pole no election means the strip club, dancing and embracing her sexuality

She is explaining that she ignores people who hate on her body because it's not for them anyway

Meg doesn't care about what people have to say about her body because her body is not for them. She is reclaiming herself.

She is reminding people that she is successful and that people hate her because of it

Defekt, biyouyumlu materyallerle kapatılmalıdır.

Etrafındaki dokularda şiddetli irritasyona neden olma.

Pulpa enfeksiyonlarının apikal foramen yoluyla apikal dokulara iletimi

Cerrahi müdahaleye gerek kalmadan tedavi sağlanma olasılığı vardır veya tedavi alveolar cerrahi ile yapılabilir.

This is an unfounded argument ; Inosine bases are perfectly fine to use in qPCR primers

testimonies should auto slide

the gradation should be like figma

The main argument by the Oneida tribe here is that all the Native American tribes need to remain neutral during the conflict between the colonists and Native Americans. The tribe also argues that both sides should not try and draw the tribes into the conflict as they aim for peace. This, however, proves to later on be hypocritical as the Oneida tribe joins forces with the colonists in the American Revolution.

Other perspectives need to be questioned - Is any of this letter by the tribe altered in any way with the interest of the missionary? How does this impact the piece?

The purpose of this document is to outline the Oneida's stance on the American Revolution, and express their middle stance between the colonists and Great Britain. They address governor and leaders of the New England colonies to set their neutrality, and urge both sides to do the same by co-existing along with each other.

Is this from all the Oneida chiefs to show the unanimous decision within the tribe? Or is this the only chiefs who agreed to neutrality?

Question; Why did the Native tribe change their mind on supporting the colonists? How long did it take them to change their mind? Upon further research, the Oneida tribe was the first Native American tribe to pledge their allegiance to the colonists, so what made them change their mind from this letter?

Question: Why do the natives refer to the leaders of New England as "chiefs"? Do they do so because they have they have a similar government set-up in the tribes and see the New England Chiefs as equals/allies within North America?

Who: The letter often refers to the metaphor of the "two brothers" with respect to the American Colonies and Great Britain. It is used to push the Native need for peace in the sense that the two Nations should co-exist with one another as they are closely related. The Natives also go onto say that "you are two brothers of one blood", relating back to the colonists roots in England and purveying that the American Colonies are closer connected to Great Britain than they wish to think.

When: This was around 2 months after the start of the American Revolution, where the first shots were fired at the battle of Lexington and Concord. This marks the point where the official conflict between the American Colonies and the British escalated, and it makes sense that the Native American tribe felt they needed to state their stance as war was nearing early America.

Who: A Native American tribe of the Iroquois who resided in central New York and later Wisconsin. They often interacted with the early northeastern colonies. Upon further research, the tribe eventually took the side of the American colonies and even fought alongside the colonies during the American Revolution. With this, they were known as "The First Ally of American" during the war.

Definition: Alien and foreign Nation is used in this context as a way to describe an outside country or force. The Natives are not using this to refer to Great Britain however, as they see the colonies and Great Britain as the need to be one.

Expressing how the Native tribes will refuse favor from both sides in an effort to remain neutral.

Definition: Announcing that they will not be choosing sides, instead remaining in the middle of the conflict between the colonies and Great Britain.

Submitted to Meta-Psychology. Participate in open peer review by sending an email to open.peer.reviewer@gmail.com. The full editorial process of all articles under review at Meta-Psychology can be found following this link:

https://tinyurl.com/mp-submissions

You will find this preprint by searching for the first author's name.

SITC continuously evaluates the field for emerging data and new FDA approvals. Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC Lung Cancer and Mesothelioma Immunotherapy Guideline Expert Panel. More information on the SITC Guidelines can be found at sitcancer.org/guidelines.

v2.1(A) Update Summary

An addendum to the SITC Lung Cancer and Mesothelioma CPG is currently in preparation to address new FDA approvals and practice-changing data for the treatment of metastatic or locally advanced NSCLC, resectable NSCLC, LS-SCLC, and malignant pleural mesothelioma using ICI-based treatment regimens. This update in preparation will also address the FDA approval of the bispecific antibody, tarlatamab, for the treatment of ES-SCLC.

This shows how we need to consider what Thomas's account offers. This account shapes readers minds on the settlers and their lifestyles. Knowing the desire to keep peace helps give a better understanding of the settlers and their relationships. Understanding the perspectives of both parties helps give a better perspective to all those involved. Knowing where they stand will help give stronger insight to those reviewing and learning history as it was and is.

This line answers the question of who had an interesting counter to predictions of settlers life style. Knowing more about Thomas and his contradiction to Robert allows for a deeper understanding of what the settlers are about to face. Knowing predictions as to what the settlers will face not only tells the reader what is occurring but also allows for a more thorough understanding. Knowing this helps shape the readers views of the settlers. Being able to predict what awaits settlers helps the readers tremendously.

Author response:

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

eLife Assessment

This important work presents a new methodology for the statistical analysis of fiber photometry data, improving statistical power while avoiding the bias inherent in the choices that are necessarily made when summarizing photometry data. The reanalysis of two recent photometry data sets, the simulations, and the mathematical detail provide convincing evidence for the utility of the method and the main conclusions, however, the discussion of the re-analyzed data is incomplete and would be improved by a deeper consideration of the limitations of the original data. In addition, consideration of other data sets and photometry methodologies including non-linear analysis tools, as well as a discussion of the importance of the data normalization are needed.

Thank you for reviewing our manuscript and giving us the opportunity to respond and improve our paper. In our revision, we have strived to address the points raised in the comments, and implement suggested changes where feasible. We have also improved our package and created an analysis guide (available on our Github - https://github.com/gloewing/fastFMM and https://github.com/gloewing/photometry_fGLMM), showing users how to apply our methods and interpret their results. Below, we provide a detailed point-by-point response to the reviewers.

Reviewer #1:

Summary:

Fiber photometry has become a very popular tool in recording neuronal activity in freely behaving animals. Despite the number of papers published with the method, as the authors rightly note, there are currently no standardized ways to analyze the data produced. Moreover, most of the data analyses confine to simple measurements of averaged activity and by doing so, erase valuable information encoded in the data. The authors offer an approach based on functional linear mixed modeling, where beyond changes in overall activity various functions of the data can also be analyzed. More in-depth analysis, more variables taken into account, and better statistical power all lead to higher quality science.

Strengths:

The framework the authors present is solid and well-explained. By reanalyzing formerly published data, the authors also further increase the significance of the proposed tool opening new avenues for reinterpreting already collected data.

Thank you for your favorable and detailed description of our work!

Weaknesses:

However, this also leads to several questions. The normalization method employed for raw fiber photometry data is different from lab to lab. This imposes a significant challenge to applying a single tool of analysis.

Thank you for these important suggestions. We agree that many data pre-processing steps will influence the statistical inference from our method. Note, though, that this would also be the case with standard analysis approaches (e.g., t-tests, correlations) applied to summary measures like AUCs. For that reason, we do not believe that variability in pre-processing is an impediment to widespread adoption of a standard analysis procedure. Rather, we would argue that the sensitivity of analysis results to pre-processing choices should motivate the development of statistical techniques that reduce the need for pre-processing, and properly account for structure in the data arising from experimental designs. For example, even without many standard pre-processing steps, FLMM provides smooth estimation results across trial timepoints (i.e., the “functional domain”), has the ability to adjust for betweentrial and -animal heterogeneity, and provides a valid statistical inference framework that quantifies the resulting uncertainty. We appreciate the reviewer’s suggestion to emphasize and further elaborate on our method from this perspective. We have now included the following in the Discussion section:

“FLMM can help model signal components unrelated to the scientific question of interest, and provides a systematic framework to quantify the additional uncertainty from those modeling choices. For example, analysts sometimes normalize data with trial-specific baselines because longitudinal experiments can induce correlation patterns across trials that standard techniques (e.g., repeated measures ANOVA) may not adequately account for. Even without many standard data pre-processing steps, FLMM provides smooth estimation results across trial time-points (the “functional domain”), has the ability to adjust for between-trial and -animal heterogeneity, and provides a valid statistical inference approach that quantifies the resulting uncertainty. For instance, session-to-session variability in signal magnitudes or dynamics (e.g., a decreasing baseline within-session from bleaching or satiation) could be accounted for, at least in part, through the inclusion of trial-level fixed or random effects. Similarly, signal heterogeneity due to subject characteristics (e.g., sex, CS+ cue identity) could be incorporated into a model through inclusion of animal-specific random effects. Inclusion of these effects would then influence the width of the confidence intervals. By expressing one’s “beliefs” in an FLMM model specification, one can compare models (e.g., with AIC). Even the level of smoothing in FLMM is largely selected as a function of the data, and is accounted for directly in the equations used to construct confidence intervals. This stands in contrast to “trying to clean up the data” with a pre-processing step that may have an unknown impact on the final statistical inferences.”

Does the method that the authors propose work similarly efficiently whether the data are normalized in a running average dF/F as it is described in the cited papers? For example, trace smoothing using running averages (Jeong et al. 2022) in itself may lead to pattern dilution.

By modeling trial signals as “functions”, the method accounts for and exploits correlation across trial timepoints and, as such, any pre-smoothing of the signals should not negatively affect the validity of the 95% CI coverage. It will, however, change inferential results and the interpretation of the data, but this is not unique to FLMM, or many other statistical procedures.

The same question applies if the z-score is calculated based on various responses or even baselines. How reliable the method is if the data are non-stationery and the baselines undergo major changes between separate trials?

Adjustment for trial-to-trial variability in signal magnitudes or dynamics could be accounted for, at least in part, through the inclusion of trial-level random effects. This heterogeneity would then influence the width of the confidence intervals, directly conveying the effect of the variability on the conclusions being drawn from the data. This stands in contrast to “trying to clean up the data” with a pre-processing step that may have an unknown impact on the final statistical inferences. Indeed, non-stationarity (e.g., a decreasing baseline within-session) due to, for example, measurement artifacts (e.g., bleaching) or behavioral causes (e.g., satiation, learning) should, if possible, be accounted for in the model. As mentioned above, one can often achieve the same goals that motivate pre-processing steps by instead applying specific FLMM models (e.g., that include trial-specific intercepts to reflect changes in baseline) to the unprocessed data. One can then compare model criteria in an objective fashion (e.g., with AIC) and quantify the uncertainty associated with those modeling choices. Even the level of smoothing in FLMM is largely selected as a function of the data, and is accounted for directly in the equations used to construct confidence intervals. In sum, our method provides both a tool to account for challenges in the data, and a systematic framework to quantify the additional uncertainty that accompanies accounting for those data characteristics.

Finally, what is the rationale for not using non-linear analysis methods? Following the paper’s logic, non-linear analysis can capture more information that is diluted by linear methods.

This is a good question that we imagine many readers will be curious about as well. We have added in notes to the Discussion and Methods Section 4.3 to address this (copied below). We thank the reviewer for raising this point, as your feedback also motivated us to discuss this point in Part 5 of our Analysis Guide.

Methods

“FLMM models each trial’s signal as a function that varies smoothly across trial time-points (i.e., along the “functional domain”). It is thus a type of non-linear modeling technique over the functional domain, since we do not assume a linear model (straight line). FLMM and other functional data analysis methods model data as functions, when there is a natural ordering (e.g., time-series data are ordered by time, imaging data are ordered by x-y coordinates), and are assumed to vary smoothly along the functional domain (e.g., one assumes values of a photometry signal at close time-points in a trial have similar values). Functional data analysis approaches exploit this smoothness and natural ordering to capture more information during estimation and inference.”

Discussion

“In this paper, we specified FLMM models with linear covariate–signal relationships at a fixed trial time-point across trials/sessions, to compare the FLMM analogue of the analyses conducted in (Jeong et al., 2022). However, our package allows modeling of covariate–signal relationships with non-linear functions of covariates, using splines or other basis functions. One must consider, however, the tradeoff between flexibility and interpretability when specifying potentially complex models, especially since FLMM is designed for statistical inference.”

Reviewer #2:

Summary:

This work describes a statistical framework that combines functional linear mixed modeling with joint 95% confidence intervals, which improves statistical power and provides less conservative statistical inferences than in previous studies. As recently reviewed by Simpson et al. (2023), linear regression analysis has been used extensively to analyze time series signals from a wide range of neuroscience recording techniques, with recent studies applying them to photometry data. The novelty of this study lies in 1) the introduction of joint 95% confidence intervals for statistical testing of functional mixed models with nested random-effects, and 2) providing an open-source R package implementing this framework. This study also highlights how summary statistics as opposed to trial-by-trial analysis can obscure or even change the direction of statistical results by reanalyzing two other studies.

Strengths:

The open-source package in R using a similar syntax as the lme4 package for the implementation of this framework on photometry data enhances the accessibility, and usage by other researchers. Moreover, the decreased fitting time of the model in comparison with a similar package on simulated data, has the potential to be more easily adopted.

The reanalysis of two studies using summary statistics on photometry data (Jeong et al., 2022; Coddington et al., 2023) highlights how trial-by-trial analysis at each time-point on the trial can reveal information obscured by averaging across trials. Furthermore, this work also exemplifies how session and subject variability can lead to opposite conclusions when not considered.

We appreciate the in-depth description of our work and, in particular, the R package. This is an area where we put a lot of effort, since our group is very concerned with the practical experience of users.

Weaknesses:

Although this work has reanalyzed previous work that used summary statistics, it does not compare with other studies that use trial-by-trial photometry data across time-points in a trial. As described by the authors, fitting pointwise linear mixed models and performing t-test and BenjaminiHochberg correction as performed in Lee et al. (2019) has some caveats. Using joint confidence intervals has the potential to improve statistical robustness, however, this is not directly shown with temporal data in this work. Furthermore, it is unclear how FLMM differs from the pointwise linear mixed modeling used in this work.

Thank you for making this important point. We agree that this offers an opportunity to showcase the advantages of FLMM over non-functional data analysis methods, such as the approach applied in Lee et al. (2019). As mentioned in the text, fitting entirely separate models at each trial timepoint (without smoothing regression coefficient point and variance estimates across timepoints), and applying multiple comparisons corrections as a function of the number of time points has substantial conceptual drawbacks. To see why, consider that applying this strategy with two different sub-sampling rates requires adjustment for different numbers of comparisons, and could thus lead to very different proportions of timepoints achieving statistical significance. In light of your comments, we decided that it would be useful to provide a demonstration of this. To that effect, we have added Appendix Section 2 comparing FLMM with the method in Lee et al. (2019) on a real dataset, and show that FLMM yields far less conservative and more stable inference across different sub-sampling rates. We conducted this comparison on the delay-length experiment (shown in Figure 6) data, sub-sampled at evenly spaced intervals at a range of sampling rates. We fit either a collection of separate linear mixed models (LMM) followed by a Benjamini–Hochberg (BH) correction, or FLMM with statistical significance determined with both Pointwise and Joint 95% CIs. As shown in Appendix Tables 1-2, the proportion of timepoints at which effects are statistically significant with FLMM Joint CIs is fairly stable across sampling rates. In contrast, the percentage is highly inconsistent with the BH approach and is often highly conservative. This illustrates a core advantage of functional data analysis methods: borrowing strength across trial timepoints (i.e., the functional domain), can improve estimation efficiency and lower sensitivity to how the data is sub-sampled. A multiple comparisons correction may, however, yield stable results if one first smooths both regression coefficient point and variance estimates. Because this includes smoothing the coefficient point and variance estimates, this approach would essentially constitute a functional mixed model estimation strategy that uses multiple comparisons correction instead of a joint CI. We have now added in a description of this experiment in Section 2.4 (copied below).

“We further analyze this dataset in Appendix Section 2, to compare FLMM with the approach applied in Lee et al. (2019) of fitting pointwise LMMs (without any smoothing) and applying a Benjamini–Hochberg (BH) correction. Our hypothesis was that the Lee et al. (2019) approach would yield substantially different analysis results, depending on the sampling rate of the signal data (since the number of tests being corrected for is determined by the sampling rate). The proportion of timepoints at which effects are deemed statistically significant by FLMM joint 95% CIs is fairly stable across sampling rates. In contrast, that proportion is both inconsistent and often low (i.e., highly conservative) across sampling rates with the Lee et al. (2019) approach. These results illustrate the advantages of modeling a trial signal as a function, and conducting estimation and inference in a manner that uses information across the entire trial.”

In this work, FLMM usages included only one or two covariates. However, in complex behavioral experiments, where variables are correlated, more than two may be needed (see Simpson et al. (2023), Engelhard et al. (2019); Blanco-Pozo et al. (2024)). It is not clear from this work, how feasible computationally would be to fit such complex models, which would also include more complex random effects.

Thank you for bringing this up, as we endeavored to create code that is able to scale to complex models and large datasets. We agree that highlighting this capability in the paper will strengthen the work. We now state in the Discussion section that “[T]he package is fast and maintains a low memory footprint even for complex models (see Section 4.6 for an example) and relatively large datasets.” Methods Section 4.6 now includes the following:

Our fastFMM package scales to the dataset sizes and model specifications common in photometry. The majority of the analyses presented in the Results Section (Section 2) included fairly simple functional fixed and random effect model specifications because we were implementing the FLMM versions of the summary measure analyses presented in Jeong et al. (2022). However, we fit the following FLMM to demonstrate the scalability of our method with more complex model specifications:

We use the same notation as the Reward Number model in Section 4.5.2, with the additional variable TL_i,j,l_ denoting the Total Licks on trial j of session l for animal i. In a dataset with over 3,200 total trials (pooled across animals), this model took ∼1.2 min to fit on a MacBook Pro with an Apple M1 Max chip with 64GB of RAM. Model fitting had a low memory footprint. This can be fit with the code:

model_fit = fui(photometry ~ session + trial + iri + lick_time + licks + (session + trial + iri + lick_time + licks | id), parallel = TRUE, data = photometry_data)

This provides a simple illustration of the scalability of our method. The code (including timing) for this demonstration is now included on our Github repository.

Reviewer #3:

Summary:

Loewinger et al., extend a previously described framework (Cui et al., 2021) to provide new methods for statistical analysis of fiber photometry data. The methodology combines functional regression with linear mixed models, allowing inference on complex study designs that are common in photometry studies. To demonstrate its utility, they reanalyze datasets from two recent fiber photometry studies into mesolimbic dopamine. Then, through simulation, they demonstrate the superiority of their approach compared to other common methods.

Strengths:

The statistical framework described provides a powerful way to analyze photometry data and potentially other similar signals. The provided package makes this methodology easy to implement and the extensively worked examples of reanalysis provide a useful guide to others on how to correctly specify models.

Modeling the entire trial (function regression) removes the need to choose appropriate summary statistics, removing the opportunity to introduce bias, for example in searching for optimal windows in which to calculate the AUC. This is demonstrated in the re-analysis of Jeong et al., 2022, in which the AUC measures presented masked important details about how the photometry signal was changing.

Meanwhile, using linear mixed methods allows for the estimation of random effects, which are an important consideration given the repeated-measures design of most photometry studies.

We would like to thank the reviewer for the deep reading and understanding of our paper and method, and the thoughtful feedback provided. We agree with this summary, and will respond in detail to all the concerns raised.

Weaknesses:

While the availability of the software package (fastFMM), the provided code, and worked examples used in the paper are undoubtedly helpful to those wanting to use these methods, some concepts could be explained more thoroughly for a general neuroscience audience.

Thank you for this point. While we went to great effort to explain things clearly, our efforts to be concise likely resulted in some lack of clarity. To address this, we have created a series of analysis guides for a more general neuroscience audience, reflecting our experience working with researchers at the NIH and the broader community. These guides walk users through the code, its deployment in typical scenarios, and the interpretation of results.

While the methodology is sound and the discussion of its benefits is good, the interpretation and discussion of the re-analyzed results are poor:

In section 2.3, the authors use FLMM to identify an instance of Simpson’s Paradox in the analysis of Jeong et al. (2022). While this phenomenon is evident in the original authors’ metrics (replotted in Figure 5A), FLMM provides a convenient method to identify these effects while illustrating the deficiencies of the original authors’ approach of concatenating a different number of sessions for each animal and ignoring potential within-session effects.

Our goal was to demonstrate that FLMM provides insight into why the opposing within- and between-session effects occur: the between-session and within-session changes appear to occur at different trial timepoints. Thus, while the AUC metrics applied in Jeong et al. (2022) are enough to show the presence of Simpson’s paradox, it is difficult to hypothesize why the opposing within-/between-session effects occur. An AUC analysis cannot determine at what trial timepoints (relative to licking) those opposing trends occur.

The discussion of this result is muddled. Having identified the paradox, there is some appropriate speculation as to what is causing these opposing effects, particularly the decrease in sessions. In the discussion and appendices, the authors identify (1) changes in satiation/habitation/motivation, (2) the predictability of the rewards (presumably by the click of a solenoid valve) and (3) photobleaching as potential explanations of the decrease within days. Having identified these effects, but without strong evidence to rule all three out, the discussion of whether RPE or ANCCR matches these results is probably moot. In particular, the hypotheses developed by Jeong et al., were for a random (unpredictable) rewards experiment, whereas the evidence points to the rewards being sometimes predictable. The learning of that predictability (e.g. over sessions) and variation in predictability (e.g. by attention level to sounds of each mouse) significantly complicate the analysis. The FLMM analysis reveals the complexity of analyzing what is apparently a straightforward task design.

While we are disappointed to hear the reviewer felt our initial interpretations and discussion were poor, the reviewer brings up an excellent point re: potential reward predictability that we had not considered. They have convinced us that acknowledging this alternative perspective will strengthen the paper, and we have added it into the Discussion. We agree that the ANCCR/RPE model predictions were made for unpredictable rewards and, as the reviewer rightly points out, there is evidence that the animals may sense the reward delivery. After discussing extensively with the authors of Jeong et al. (2022), it is clear that they went to enormous trouble to prevent the inadvertent generation of a CS+, and it is likely changes in pressure from the solenoid (rather than a sound) that may have served as a cue. Regardless of the learning theory one adopts (RPE, ANCCR or others), we agree that this potential learned predictability could, at least partially, account for the increase in signal magnitude across sessions. As this paper is focused on analysis methods, we feel that we can contribute most thoughtfully to the dopamine–learning theory conversation by presenting this explanation in detail, for consideration in future experiments. We have substantially edited this discussion and, as per the reviewer’s suggestion, have qualified our interpretations to reflect the uncertainty in explaining the observed trends.

If this paper is not trying to arbitrate between RPE and ANCCR, as stated in the text, the post hoc reasoning of the authors of Jeong et al 2022 provided in the discussion is not germane. Arbitrating between the models likely requires new experimental designs (removing the sound of the solenoid, satiety controls) or more complex models (e.g. with session effects, measures of predictability) that address the identified issues.

Thank you for this point. We agree with you that, given the scope of the paper, we should avoid any extensive comparison between the models. To address your comment, we have now removed portions of the Discussion that compared RPE and ANCCR. Overall, we agree with the reviewer, and think that future experiments will be needed for conclusively testing the accuracy of the models’ predictions for random (unpredicted) rewards. While we understand that our description of several conversations with the Jeong et al., 2022 authors could have gone deeper, we hope the reviewer can appreciate that inclusion of these conversations was done with the best of intentions. We wish to emphasize that we also consulted with several other researchers in the field when crafting our discussion. We do commend the authors of Jeong et al., 2022 for their willingness to discuss all these details. They could easily have avoided acknowledging any potential incompleteness of their theory by claiming that our results do not invalidate their predictions for a random reward, because the reward could potentially have been predicted (due to an inadvertent CS+ generated from the solenoid pressure). Instead, they emphasized that they thought their experiment did test a random reward, to the extent they could determine, and that our results suggest components of their theory that should be updated. We think that engagement with re-analyses of one’s data, even when findings are at odds with an initial theoretical framing, is a good demonstration of open science practice. For that reason as well, we feel providing readers with a perspective on the entire discussion will contribute to the scientific discourse in this area.

Finally, we would like to reiterate that this conversation is happening at least in part because of our method: by analyzing the signal at every trial timepoint, it provides a formal way to test for the presence of a neural signal indicative of reward delivery perception. Ultimately, this was what we set out to do: help researchers ask questions of their data that may have been harder to ask before. We believe that having a demonstration that we can indeed do this for a “live” scientific issue is the most appropriate way of demonstrating the usefulness of the method.

Of the three potential causes of within-session decreases, the photobleaching arguments advanced in the discussion and expanded greatly in the appendices are not convincing. The data being modeled is a processed signal (∆F/F) with smoothing and baseline correction and this does not seem to have been considered in the argument. Furthermore, the photometry readout is also a convolution of the actual concentration changes over time, influenced by the on-off kinetics of the sensor, which makes the interpretation of timing effects of photobleaching less obvious than presented here and more complex than the dyes considered in the cited reference used as a foundation for this line of reasoning.

We appreciate the nuance of this point, and we have made considerable efforts in the Results and Discussion sections to caution that alternative hypotheses (e.g., photobleaching) cannot be definitively ruled out. In response to your criticism, we have consulted with more experts in the field regarding the potential for bleaching in this data, and it is not clear to us why photobleaching would be visible in one time-window of a trial, but not at another (less than a second away), despite high ∆F/F magnitudes in both time-windows. We do wish to point out that the Jeong et al. (2022) authors were also concerned about photobleaching as a possible explanation. At their request, we analyzed data from additional experiments, collected from the same animals. In most cases, we did not observe signal patterns that seemed to indicate photobleaching. Given the additional scrutiny, we do not think that photobleaching is more likely to invalidate results in this particular set of experiments than it would be in any other photometry experiment. While the role of photobleaching may be more complicated with this sensor than others in the references, that citation was included primarily as a way of acknowledging that it is possible that non-linearities in photobleaching could occur. Regardless, your point is well taken and we have qualified our description of these analyses to express that photobleaching cannot be ruled out.

Within this discussion of photobleaching, the characterization of the background reward experiments used in part to consider photobleaching (appendix 7.3.2) is incorrect. In this experiment (Jeong et al., 2022), background rewards were only delivered in the inter-trial-interval (i.e. not between the CS+ and predicted reward as stated in the text). Both in the authors’ description and in the data, there is a 6s before cue onset where rewards are not delivered and while not described in the text, the data suggests there is a period after a predicted reward when background rewards are not delivered. This complicates the comparison of this data to the random reward experiment.

Thank you for pointing this out! We removed the parenthetical on page 18 of the appendix that incorrectly stated that rewards can occur between the CS+ and the predicted reward.

The discussion of the lack of evidence for backpropagation, taken as evidence for ANCCR over RPE, is also weak.

Our point was initially included to acknowledge that, although our method yields results that conflict with the conclusions described by Jeong et al., 2022 on data from some experiments, on other experiments our method supports their results. Again, we believe that a critical part of re-analyzing shared datasets is acknowledging both areas where new analyses support the original results, as well as those where they conflict with them. We agree with the reviewer that qualifying our results so as not to emphasize support for/against RPE/ANCCR will strengthen our paper, and we have made those changes. We have qualified the conclusions of our analysis to emphasize they are a demonstration of how FLMM can be used to answer a certain style of question with hypothesis testing (how signal dynamics change across sessions), as opposed to providing evidence for/against the backpropagation hypothesis.

A more useful exercise than comparing FLMM to the methods and data of Jeong et al., 2022, would be to compare against the approach of Amo et al., 2022, which identifies backpropagation (data publicly available: DOI: 10.5061/dryad.hhmgqnkjw). The replication of a positive result would be more convincing of the sensitivity of the methodology than the replication of a negative result, which could be a result of many factors in the experimental design. Given that the Amo et al. analysis relies on identifying systematic changes in the timing of a signal over time, this would be particularly useful in understanding if the smoothing steps in FLMM obscure such changes.

Thank you for this suggestion. Your thoughtful review has convinced us that focusing on our statistical contribution will strengthen the paper, and we made changes to further emphasize that we are not seeking to adjudicate between RPE/ANCCR. Given the length of the manuscript as it stands, we could only include a subset of the analyses conducted on Jeong et al., 2022, and had to relegate the results from the Coddington et al., data to an appendix. Realistically, it would be hard for us to justify including analyses from a third dataset, only to have to relegate them to an appendix. We did include numerous examples in our manuscript where we already replicated positive results, in a way that we believe demonstrates the sensitivity of the methodology. We have also been working with many groups at NIH and elsewhere using our approach, in experiments targeting different scientific questions. In fact, one paper that extensively applies our method, and compares the results with those yielded by standard analysis of AUCs, is already published (Beas et al., 2024). Finally, in our analysis guide we describe additional analyses, not included in the manuscript, that replicate positive results. Hence there are numerous demonstrations of FLMM’s performance in less controversial settings. We take your point that our description of the data supporting one theory or the other should be qualified, and we have corrected that. Specifically for your suggestion of Amo et al. 2022, we have not had the opportunity to personally reanalyze their data, but we are already in contact with other groups who have conducted preliminary analyses of their data with FLMM. We are delighted to see this, in light of your comments and our decision to restrict the scope of our paper. We will help them and other groups working on this question to the extent we can.

Recommendations for the Authors:

Reviewer #2:

First, I would like to commend the authors for the clarity of the paper, and for creating an open-source package that will help researchers more easily adopt this type of analysis.

Thank you for the positive feedback!

I would suggest the authors consider adding to the manuscript, either some evidence or some intuition on how feasible would be to use FLMM for very complex model specifications, in terms of computational cost and model convergence.

Thank you for this suggestion. As we described above in response to Reviewer #2’s Public Reviews, we have added in a demonstration of the scalability of the method. Since our initial manuscript submission, we have further increased the package’s speed (e.g., through further parallelization). We are releasing the updated version of our package on CRAN.

From my understanding, this package might potentially be useful not just for photometry data but also for two-photon recordings for example. If so, I would also suggest the authors add to the discussion this potential use.

This is a great point. Our updated manuscript Discussion includes the following:

“The FLMM framework may also be applicable to techniques like electrophysiology and calcium imaging. For example, our package can fit functional generalized LMMs with a count distribution (e.g., Poisson). Additionally, our method can be extended to model time-varying covariates. This would enable one to estimate how the level of association between signals, simultaneously recorded from different brain regions, fluctuates across trial time-points. This would also enable modeling of trials that differ in length due to, for example, variable behavioral response times (e.g., latency-topress).”

Reviewer #3:

The authors should define ’function’ in context, as well as provide greater detail of the alternate tests that FLMM is compared to in Figure 7.

We include a description of the alternate tests in Appendix Section 5.2. We have updated the Methods Section (Section 4) to introduce the reader to how ‘functions’ are conceptualized and modeled in the functional data analysis literature. Specifically, we added the following text:

“FLMM models each trial’s signal as a function that varies smoothly across trial time-points (i.e., along the “functional domain”). It is thus a type of non-linear modeling technique over the functional domain, since we do not assume a linear model (straight line). FLMM and other functional data analysis methods model data as functions, when there is a natural ordering (e.g., time-series data are ordered by time, imaging data are ordered by x-y coordinates), and are assumed to vary smoothly along the functional domain (e.g., one assumes values of a photometry signal at close time-points in a trial have similar values). Functional data analysis approaches exploit this smoothness and natural ordering to capture more information during estimation and inference.”

Given the novelty of estimating joint CIs, the authors should be clearer about how this should be reported and how this differs from pointwise CIs (and how this has been done in the past).

We appreciate your pointing this out, as the distinction is nuanced. Our manuscript includes a description of how joint CIs enable one to interpret effects as statistically significant for time-intervals as opposed to individual timepoints. Unlike joint CIs, assessing significance with pointwise CIs suffers from multiple-comparisons problems. As a result of your suggestion, we have included a short discussion of this to our analysis guide (Part 1), entitled “Pointwise or Joint 95% Confidence Intervals.” The Methods section of our manuscript also includes the following:

“The construction of joint CIs in the context of functional data analysis is an important research question; see Cui et al. (2021) and references therein. Each point at which the pointwise 95% CI does not contain 0 indicates that the coefficient is statistically significantly different from 0 at that point. Compared with pointwise CIs, joint CIs takes into account the autocorrelation of signal values across trial time-points (the functional domain). Therefore, instead of interpreting results at a specific timepoint, joint CIs enable joint interpretations at multiple locations along the functional domain. This aligns with interpreting covariate effects on the photometry signals across time-intervals (e.g., a cue period) as opposed to at a single trial time-point. Previous methodological work has provided functional mixed model implementations for either joint 95% CIs for simple random-effects models (Cui et al., 2021), or pointwise 95% CIs for nested models (Scheipl et al., 2016), but to our knowledge, do not provide explicit formulas or software for computing joint 95% CIs in the presence of general random-effects specifications.”

The authors identify that many photometry studies are complex nested longitudinal designs, using the cohort of 8 animals used in five task designs of Jeong et al. 2022 as an example. The authors miss the opportunity to illustrate how FLMM might be useful in identifying the effects of subject characteristics (e.g. sex, CS+ cue identity).

This is a fantastic point and we have added the following into the Discussion:

“...[S]ignal heterogeneity due to subject characteristics (e.g., sex, CS+ cue identity) could be incorporated into a model through inclusion of animal-specific random effects.”

In discussing the delay-length change experiment, it would be more accurate to say that proposed versions of RPE and ANCCR do not predict the specific change.

Good point. We have made this change.

Minor corrections:

Panels are mislabeled in Figure 5.

Thank you. We have corrected this.

The Crowder (2009) reference is incorrect, being a review of the book with the book presumably being the correct citation.

Good catch, thank you! Corrected.

In Section 5 (first appendix), the authors could include the alternate spelling ’fibre photometry’ to capture any citations that use British English spelling.

This is a great suggestion, but we did not have time to recreate these figures before re-submission.

Section 7.4 is almost all quotation, though unevenly using the block quotation formatting. It is unclear why such a large quotation is included.

Thank you for pointing this out. We have removed this Appendix section (formerly Section 7.4) as the relevant text was already included in the Methods section.

References

Sofia Beas, Isbah Khan, Claire Gao, Gabriel Loewinger, Emma Macdonald, Alison Bashford, Shakira Rodriguez-Gonzalez, Francisco Pereira, and Mario A Penzo. Dissociable encoding of motivated behavior by parallel thalamo-striatal projections. Current Biology, 34(7):1549–1560, 2024.

Erjia Cui, Andrew Leroux, Ekaterina Smirnova, and Ciprian Crainiceanu. Fast univariate inference for longitudinal functional models. Journal of Computational and Graphical Statistics, 31:1–27, 07 2021. doi: 10.1080/10618600.2021.1950006.

Huijeong Jeong, Annie Taylor, Joseph R Floeder, Martin Lohmann, Stefan Mihalas, Brenda Wu, Mingkang Zhou, Dennis A Burke, and Vijay Mohan K Namboodiri. Mesolimbic dopamine release conveys causal associations. Science, 378(6626):eabq6740, 2022. doi: 10.1126/science.abq6740. URL https://www. science.org/doi/abs/10.1126/science.abq6740.

Rachel S Lee, Marcelo G Mattar, Nathan F Parker, Ilana B Witten, and Nathaniel D Daw. Reward prediction error does not explain movement selectivity in dms-projecting dopamine neurons. eLife, 8:e42992, apr 2019. ISSN 2050-084X. doi: 10.7554/eLife.42992. URL https://doi.org/10.7554/eLife.42992.

Fabian Scheipl, Jan Gertheiss, and Sonja Greven. Generalized functional additive mixed models. Electronic Journal of Statistics, 10(1):1455 – 1492, 2016. doi: 10.1214/16-EJS1145. URL https://doi.org/10.1214/16-EJS1145.

eLife Assessment

This important study presents a statistical framework for the analysis of photometry signals and provides an open-source implementation. The evidence supporting the benefits of the presented functional mixed-effect modeling analysis as opposed to 1) summary statistics and 2) other pointwise regression models is convincing with a thorough comparison with other methods and datasets. This work will be of great interest to researchers using not only fiber photometry, but other time-series data such as calcium imaging or electrophysiology data, and wanting to implement trial-by-trial temporal analysis, taking also into account variability within the dataset.

Reviewer #1 (Public review):

Summary:

Fiber photometry has become a very popular tool in recording neuronal activity in freely behaving animals. Despite the number of papers published with the method, as the authors rightly note, there are currently no standardized ways to analyze the data produced. Moreover, most of the data analyses confine to simple measurements of averaged activity and by doing so, erase valuable information encoded in the data. The authors offer an approach based on functional linear mixed modeling, where beyond changes in overall activity various functions of the data can also be analyzed. More in depth analysis, more variables taken into account, better statistical power all lead to higher quality science.

Strengths:

The framework the authors present is solid and well explained. By reanalyzing formerly published data, the authors also further increase the significance of the proposed tool opening new avenues for reinterpreting already collected data. They also made a convincing case showing that the proposed algorithm works on data with different preprocessing backgrounds.

testing annotation

1. Global cooperation incentives: Find incentives that motivate countries and MNCs to cooperate and act pro-socially.
2. Identify and promote efficient structures of cooperation on various levels (international, national, township, family etc.)
3. How to alleviate the suffering of the poorest

@emilio Not aligned with the Google Doc.

This sshould be configured as a mailt o link that opens the user's default email client when clicked, directing them to send an email to hello@edm8ker.com.

This button should be configured as a mailto link that opens the user's default email client when clicked, directing them to send an email to hello@edm8ker.com.

The section after this point is missing. @emilio ref to https://docs.google.com/document/d/1RNKQyGQX3xPUyXYuKRIoPodcYTnDQHCrh2kn2cPf19c/edit

eLife Assessment

This article presents valuable findings on the impact of climate change on odonates, integrating phenological and range shifts to broaden our understanding of biodiversity change. The study leverages extensive natural history data, offering a combined analysis of temporal trends in phenology and distribution and their potential drivers. The support for the findings is solid, though additional clarification regarding the methods and alternative sensitivity analyses could make the conclusions stronger.

The headline is just "Why edm8ker?"

The points aren't in order of preference. Ref: figma Also, it's just "People"

It should be on the next line.

eLife Assessment

This study poses an important step forward in understanding the brain-network embedding of beta oscillations. The study advances our circuit-level understanding of the pathophysiology associated with dopaminergic alterations in psychiatric or neurological disorders. The study provides compelling evidence that beta oscillations across the neocortex and basal ganglia map onto shared functional and structural networks that show significant positive correlations with dopamine receptors.

Reviewer #1 (Public review):

The study by Chikermane and colleagues investigates functional, structural, and dopaminergic network substrate of cortical beta oscillations (13-30 Hz). The major strength of the work lies in the methodology taken by the authors, namely a multimodal lesion network mapping. First, using invasive electrophysiological recordings from healthy cortical territories of epileptic patients they identify regions with highest beta power. Next, they leverage open access MRI data and PET atlases and use the identified high-beta regions as seeds to find (1) the whole-brain functional and structural maps of regions that form the putative underlying network of high-beta regions and (2) the spatial distribution of dopaminergic receptors that show correlation with nodal connectivity of the identified networks. These steps are achieved by generating aggregate functional, structural, and dopaminergic network maps using lead-DBS toolbox, and by contrasting the results with those obtained from high-alpha regions. The main findings are:

(1) Beta power is strongest across frontal, cingulate, and insular regions in invasive electrophysiological data, and these regions map onto a shared functional and structural network.<br /> (2) The shared functional and structural networks show significant positive correlations with dopamine receptors across cortex and basal ganglia (which is not the case for alpha, where correlations are found with GABA).

Reviewer #2 (Public review):

Summary:

This is a very interesting paper that leveraged several publicly available datasets: invasive cortical recording in epilepsy patients, functional and structural connectomic data, and PET data related to dopaminergic and gaba-ergic synapses. These were combined to create a unified hypothesis of beta band oscillatory activity in the human brain. They show that beta frequency activity is ubiquitous, and does not just occur in sensorimotor areas. Cortical regions where beta oscillations predominated had high connectivity to regions that are high in dopamine re-update.

Strengths:

The authors leverage and integrate three publicly available human brain datasets in a creative way. These public datasets are powerful tools for human neuroscience, and it is innovative to combine these three types of data into a common brain space to generate novel findings and hypotheses. Findings are nicely controlled by separately examining cortical regions where alpha predominates (which have a different connectivity pattern). GABA uptake from PET studies is used as a control for the specificity of the relationship between beta activity and dopamine uptake. There is much interest in synchronized oscillatory activity as a mechanism of brain function and dysfunction, but the field is short on unifying hypotheses of why particular rhythms predominate in particular regions. This paper contributes nicely to that gap. It is ambitious in generating hypotheses, particularly that modulation of beta activity may be used as a "proxy" for modulating phasic dopamine release.

Weaknesses:

As the authors point out, the use of normative data is excellent for exploring hypotheses but does not address or explore individual variations which could lead to other insights. It is also biased to resting state activity; maps of task related activity (if they were available) might show different findings.

Challenges:

In the Discussion, the authors do a fairly deep dive into the implications of their findings, particularly with respect to the hypothesis that beta band activity "preserves the status quo", and with respect to the use of beta band activity in controlling brain-machine interfaces. Mechanistically and theoretically oriented readers might gain rewarding new insights by a careful read of the discussion, but full appreciation of their deep dive may require real time interaction with the authors.

Reviewer #3 (Public review):

Summary:

In this paper, Chikermane et al. leverage a large open dataset of intracranial recordings (sEEG or ECoG) to analyze resting state (eyes closed) oscillatory activity from a variety of human brain areas. The authors identify a dominant proportion of channels in which beta band activity (12-30Hz) is most prominent, and subsequently seek to relate this to anatomical connectivity data by using the sEEG/ECoG electrodes as seeds in a large set of MRI data from the human connectome project. This reveals separate regions and white matter tracts for alpha (primarily occipital) and beta (prefrontal cortex and basal ganglia) oscillations. Finally, using a third available dataset of PET imaging, the authors relate the parcellated signals to dopamine signaling as estimated by spatial uptake patterns of dopamine, and reveal a significant correlation between the functional connectivity maps and the dopamine reuptake maps, suggesting a functional relationship between the two.

Strengths:

Overall, I found the paper well justified, focused on an important topic and interesting. The authors' use of 3 different open datasets was creative and informative, and it significantly adds to our understanding of different oscillatory networks in the human brain, and their more elusive relation with neuromodulator signaling networks by adding to our knowledge of the association between beta oscillations and dopamine signaling. Even my main comments about the lack of a theta network analysis and discussion points are relatively minor, and I believe this paper is valuable and informative.

Weaknesses:

The analyses were adequate, and the authors cleverly leverage these different datasets to build an interesting story. The main aspect I found missing (in addition to some discussion items, see below) was an examination of the theta network. Theta oscillations have been involved in a number of cognitive processes including spatial navigation and memory, and have been proposed to have different potential originating brain regions, and it would be informative to see how their anatomical networks (e.g. as in Fig. 2) look like under the author's analyses.

The authors devote a significant portion of the discussion to relating their findings to a popular hypothesis for the function of beta oscillations, the maintenance of the "status quo", mostly in the context of motor control. As the authors acknowledge, given the static nature of the data and lack of behavior, this interpretation remains largely speculative and I found it a bit too far-reaching given the data shown in the paper. In contrast, I missed a more detailed discussion on the growing literature indicating a role for beta in mood (e.g. in Kirkby et al. 2018), especially given the apparent lack of hippocampal and amygdala involvement in the paper, which was surprising.

Sería muy interesante. No me gustan los iconos y me gusta cómo se ven las aplicaciones en Dmenu y Rofi.

@emilio (Ref to google doc: https://docs.google.com/document/d/18HtK5PbMVasJpUlBrFwmNHxsX7VhihJ65Sh9xuTGEM8/edit#heading=h.7mou8kj9rv0h) This is the updated section - 4.4.5 Your Classroom's One-Stop Maker Shop Discover a treasure trove of tools, activities, and inspiration to elevate your maker education journey. From engaging blog posts and free STEM challenges to expert guidance and community support, edm8ker has everything you need to foster creativity and innovation in your classroom. CTA Button: Explore Our Resources Now!

@emilio There's one line that's missing and CTA Button content is different.

The Description is - Don’t toss that broken appliance—repair it! @emilio @arif

@emilio @Arif Name of the programme is "Future-ready Maker Programs".

The other programs have not beed added. Ref: Figma

The link is broken.

Our career quiz is no longer available.

Source: https://80000hours.org/career-quiz/

On the other hand, this situation creates an unusual diversity within a church that typically aims for consistency, especially in how services are conducted. The church’s limited guidelines for producing online Masses—seen in the lack of protocols from dioceses or bishops’ conferences—suggests a tendency to overlook this diversity.

circumscribed = restricted but in st. paul parish priest has authority to change the prayers..

definition of adjudication accordinf to what is just.

The CTA content and location were edited but aren’t showing up in Figma. Astitva will need to update this in the design.

CTA Button missing at the bottom of the section. Ref: Figma

NOTE FOR THE IMAGE: Please include images of makerspaces designed by us/ or actual makerspace. @Terence, could you let me know where I can find these photos? @Astitva can you take this up pls?

This comment answers the question of who fashioned the complex societies. This is significant as it is important to know who is doing what. Without knowing that British colonists fashioned complex societies it is harder to understand the lesson. Knowing the British formed tough societies makes it easier to know what their religion and economic ties were like. The complexity of their society is what makes their style of life so unique.

find the name for the field of study pertaining to answering the question

What's in a name?

This answers the question of what other accounts do we need to consider. While it is really important to think about the Native perspectives it is also important to consider what White might have been thinking. In this line we learn where White's perspective comes from and what shapes it. Knowing this can help up be more open to hearing from White. White gains his perspective from archaeological evidence.

Folders were developed to model trees and other hierarchical structures, not the other way around. These were all informed by hierarchical societies that humans have lived in, where king or chief has a family and thus a tree of succession

"pertaining to, performed by, or originating in the intellect," 1650s, from Greek noētikos "intelligent," from noēsis "a perception, intelligence, thought" (see noesis). Related: Noetical (1640s).

what the story exactly is

eLife Assessment

This important study substantially advances our understanding of nocturnal animal navigation and the ways that animals use polarized light. The evidence supporting the conclusions is convincing, with elegant behavioural experiments in actively navigating ants. The work will be of interest to biologists working on animal navigation or sensory ecology.

Reviewer #1 (Public review):

Freas et al. investigated if the exceedingly dim polarization pattern produced by the moon can be used by animal to guide a genuine navigational task. The sun and moon are celestial beacons for directional information, but they can be obscured by clouds, canopy, or the horizon. However, even when hidden from view, these celestial bodies provide directional information through the polarized light patterns in the sky. While the sun's polarization pattern is famously used by many animals for compass orientation, until now it has never been shown that the extremely dim polarization pattern of the moon can be used for navigation. To test this, Freas et al. studied nocturnal bull ants, by placing a linear polarizer in the homing path on a freely navigating ant 45 degrees shifted to the moon's natural polarization pattern. They recorded the homing direction of an ant before entering the polarizer, under the polarizer, and again after leaving the area covered by the polarizer. The results very clearly show, that ants walking under the linear polarizer change their homing direction by about 45 degrees in comparison to the homing direction under the natural polarization pattern and change it back after leaving the area covered by the polarizer again. These results can be repeated throughout the lunar month, showing that bull ants can use the moon's polarization pattern even under crescent moon conditions. Finally, the authors show, that the degree in which the ants change their homing direction is dependent on the length of their home vector, just as it is for the solar polarization pattern.

The behavioral experiments are very well designed, and the statistical analyses are appropriate for the data presented. The authors' conclusions are nicely supported by the data and clearly show nocturnal bull ants use the dim polarization pattern of the moon for homing, in the same way many animals use the sun's polarization pattern during the day. This is the first proof of the use of the lunar polarization pattern in any animal.

Comments on revised version:

The authors have addressed all of my previous comments and suggestions. I am happy with the way the manuscript has improved and have no further comments.

Reviewer #2 (Public review):

Summary:

The authors aimed to understand whether polarised moonlight could be used as a directional cue for nocturnal animals homing at night, particularly at times of night when polarised light is not available from the sun. To do this, the authors used nocturnal ants, and previously established methods, to show that the walking paths of ants can be altered predictably when the angle of polarised moonlight illuminating them from above is turned by a known angle (here +/- 45 degrees).

Strengths:

The behavioural data are very clear and unambiguous. The results clearly show that when the angle of downwelling polarised moonlight is turned, ants turn in the same direction. The data also clearly show that this result is maintained even for different phases (and intensities) of the moon, although during the waning cycle of the moon the ants' turn is considerably less than may be expected.

Impact:

The authors have discovered that nocturnal bull ants, while homing back to their nest holes at night, are able to use the dim polarised light pattern formed around the moon for path integration. Even though similar methods have previously shown the ability of dung beetles to orient along straight trajectories for short distances using polarised moonlight, this the first evidence of an animal that uses polarised moonlight in homing. This is quite significant, and their findings are well supported by their data.

Comments on revised version:

The authors have made a good effort to accommodate my suggestions for improvement (and from what I can tell, those of the other reviewers). I have no further comments.

Reviewer #3 (Public review):

Summary:

This manuscript presents a series of experiments aimed at investigating orientation to polarized lunar skylight in a nocturnal ant, the first report of its kind that I am aware of.

Strengths:

The study was conducted carefully and is clearly explained here.

Comments on revised version:

The manuscript is much improved and will make an excellent contribution to the field.

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Freas et al. investigated if the exceedingly dim polarization pattern produced by the moon can be used by animals to guide a genuine navigational task. The sun and moon have long been celestial beacons for directional information, but they can be obscured by clouds, canopy, or the horizon. However, even when hidden from view, these celestial bodies provide directional information through the polarized light patterns in the sky. While the sun's polarization pattern is famously used by many animals for compass orientation, until now it has never been shown that the extremely dim polarization pattern of the moon can be used for navigation. To test this, Freas et al. studied nocturnal bull ants, by placing a linear polarizer in the homing path on freely navigating ants 45 degrees shifted to the moon's natural polarization pattern. They recorded the homing direction of an ant before entering the polarizer, under the polarizer, and again after leaving the area covered by the polarizer. The results very clearly show, that ants walking under the linear polarizer change their homing direction by about 45 degrees in comparison to the homing direction under the natural polarization pattern and change it back after leaving the area covered by the polarizer again. These results can be repeated throughout the lunar month, showing that bull ants can use the moon's polarization pattern even under crescent moon conditions. Finally, the authors show, that the degree in which the ants change their homing direction is dependent on the length of their home vector, just as it is for the solar polarization pattern. 

The behavioral experiments are very well designed, and the statistical analyses are appropriate for the data presented. The authors' conclusions are nicely supported by the data and clearly show that nocturnal bull ants use the dim polarization pattern of the moon for homing, in the same way many animals use the sun's polarization pattern during the day. This is the first proof of the use of the lunar polarization pattern in any animal.

Reviewer #2 (Public Review): 

Summary: 

The authors aimed to understand whether polarised moonlight could be used as a directional cue for nocturnal animals homing at night, particularly at times of night when polarised light is not available from the sun. To do this, the authors used nocturnal ants, and previously established methods, to show that the walking paths of ants can be altered predictably when the angle of polarised moonlight illuminating them from above is turned by a known angle (here +/- 45 degrees).

Strengths: 

The behavioural data are very clear and unambiguous. The results clearly show that when the angle of downwelling polarised moonlight is turned, ants turn in the same direction. The data also clearly show that this result is maintained even for different phases (and intensities) of the moon, although during the waning cycle of the moon the ants' turn is considerably less than may be expected.

Weaknesses: 

The final section of the results - concerning the weighting of polarised light cues into the path integrator - lacks clarity and should be reworked and expanded in both the Methods and the Results (also possibly with an extra methods figure). I was really unsure of what these experiments were trying to show or what the meaning of the results actually are.

Rewrote these sections and added figure panel to Figure 6.

Impact: 

The authors have discovered that nocturnal bull ants while homing back to their nest holes at night, are able to use the dim polarised light pattern formed around the moon for path integration. Even though similar methods have previously shown the ability of dung beetles to orient along straight trajectories for short distances using polarised moonlight, this is the first evidence of an animal that uses polarised moonlight in homing. This is quite significant, and their findings are well supported by their data.

Reviewer #3 (Public Review): 

Summary: 

This manuscript presents a series of experiments aimed at investigating orientation to polarized lunar skylight in a nocturnal ant, the first report of its kind that I am aware of.

Strengths: 

The study was conducted carefully and is clearly explained here. 

Weaknesses: 

I have only a few comments and suggestions, that I hope will make the manuscript clearer and easier to understand.

Time compensation or periodic snapshots 

In the introduction, the authors compare their discovery with that in dung beetles, which have only been observed to use lunar skylight to hold their course, not to travel to a specific location as the ants must. It is not entirely clear from the discussion whether the authors are suggesting that the ants navigate home by using a time-compensated lunar compass, or that they update their polarization compass with reference to other cues as the pattern of lunar skylight gradually shifts over the course of the night - though in the discussion they appear to lean towards the latter without addressing the former. Any clues in this direction might help us understand how ants adapted to navigate using solar skylight polarization might adapt use to lunar skylight polarization and account for its different schedule. I would guess that the waxing and waning moon data can be interpreted to this effect.

Added a paragraph discussing this distinction in mechanisms and the limits of the current data set in untangling them. An interesting topic for a follow up to be sure.

Effects of moon fullness and phase on precision 

As well as the noted effect on shift magnitudes, the distributions of exit headings and reorientations also appear to differ in their precision (i.e., mean vector length) across moon phases, with somewhat shorter vectors for smaller fractions of the moon illuminated. Although these distributions are a composite of the two distributions of angles subtracted from one another to obtain these turn angles, the precision of the resulting distribution should be proportional to the original distributions. It would be interesting to know whether these differences result from poorer overall orientation precision, or more variability in reorientation, on quarter moon and crescent moon nights, and to what extent this might be attributed to sky brightness or degree of polarization.

See below for response to this and the next reviewer comment

N.B. The Watson-Williams tests for difference in mean angle are also sensitive to differences in sample variance. This can be ruled out with another variety of the test, also proposed by Watson and Williams, to check for unequal variances, for which the F statistic is = (n2-1)*(n1-R1) / (n1-1)*(n2-R2) or its inverse, whichever is >1. 

We have looked at the amount of variance from the mean heading direction in terms of both the shifts and the reorientations and found no significant difference in variance between all relevant conditions. It is possible (and probably likely) that with a higher n we might find these differences but with the current data set we cannot make statistical statements regarding degradations in navigational precision.  

As an additional analysis to address the Watson-Williams test‘s sensitivity to changes in variance, we have added var test comparisons for each of the comparisons, which is a well-established test to compare variance changes. None of these were significantly different, suggesting the observed differences in the WW tests are due to changes in the mean vector and not the distribution. We have added this test to the text.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

I have only very few minor suggestions to improve the manuscript: 

(1) While I fully agree with the authors that their study, to the best of my knowledge, provides the first proof (in any animal) of the use of the moon's polarization pattern, the many repetitions of this fact disturb the flow of the text and could be cut at several instances. 

Yes, it is indeed repeated to an annoying degree. 

We have removed these beyond bookending mentions (Abstract and Discussion).

(2) In my opinion, the authors did not change the "ambient polarization pattern" when using the linear polarization filter (e.g., l. 55, 170, 177 ...). The linear polarizer presents an artificial polarization pattern with a much higher degree of polarization in comparison to the ambient polarization pattern. I would suggest re-phrasing this, to emphasize the artificial nature of the polarization pattern under the polarizer.

We have made these suggested changes throughout the text to clarify. We no longer say the ambient pattern was   

(3) Line 377: I do not see the link between the sentence and Figure 7 

Changed where in the discussion we refer to Figure 7.

(4) Figure 7 upper part: In my opinion, the upper part of Figure 7 does not add any additional value to the illustration of the data as compared to Figure 5 and could be cut.

We thought it might be easier for some reader to see the shifts as a dial representation with the shift magnitude converted to 0-100% rather than the shifts in Figure 5. This makes it somewhat like a graphical abstract summarising the whole study.

I agree that Figure 5 tells the same story but a reader that has little background in directional stats might find figure 7 more intuitive. This was the intent at least. 

If it becomes a sticking point, then we can remove the upper portion.  

Reviewer #2 (Recommendations For The Authors): 

MINOR CORRECTIONS AND QUERIES 

Line 117: THE majority 

Corrected

Lines 129-130: Do you have a reference to support this statement? I am unaware of experiments that show that homing ants count their steps, but I could have missed it.

We have added the references that unpack the ant pedometer.  

Line 140: remove "the" in this line. 

Removed

Line 170: We need more details here about the spectral transmission properties of the polariser (and indeed which brand of filter, etc.). For instance, does it allow the transmission of UV light?

Added

Line 239: "...tested identicALLY to ...." 

Corrected

Lines 242-258 (Vector testing): I must admit I found the description of these experiments very difficult to follow. I read this section several times and felt no wiser as a result. I think some thought needs to be given to better introduce the reader to the rationale behind the experiment (e.g., start by expanding lines 243-246, and maybe add a methods figure that shows the different experimental procedures).

I have rewritten this section of the methods to clearly state the experiment rational and to be clearer as to the methodology.

Also Added a methods panel to Figure 6.

Line 247: "reoriented only halfway". What does this mean? Do you mean with half the expected angle?

Yes, this is a bit unclear. We have altered for clarity:

‘only altered their headings by about half of the 45° e-vector shift (25.2°± 3.7°), despite being tested on near-full-moon nights.’

Results section (in general): In Figure 1 (which is a very nice figure!) you go to all the trouble of defining b degrees (exit headings) and c degrees (reorientation headings), which are very intuitive for interpreting the results, and then you totally abandon these convenient angles in favour of an amorphous Greek symbol Phi (Figs. 2-6) to describe BOTH exit and reorientation headings. Why?? It becomes even more confusing when headings described by Phi can be typically greater than 300 degrees in the figures, but they are never even close to this in the text (where you seem to have gone back to using the b degrees and c degrees angles, without explicitly saying so). Personally, I think the b degrees and c degrees angles are more intuitive (and should be used in both the text and the figures), but if you do insist on using Phi then you should use it consistently in both the text and the figures. 

Replaced Phi with b° and c° for both figures and in the text.

Finally, for reorientation angles in Figure 4A, you say that the angle is 16.5 degrees. This angle should have been 143.5 degrees to be consistent with other figures. 

Yes, the reorientation was erroneously copied from the shift data (it is identical in both the +45 shift and reorientation for Figure 4A). This has now been corrected

Line 280, and many other lines: Wherever you refer to two panels of the same figure, they should be written as (say) Figure 2A, B not Figure 2AB.

Changed as requested throughout the text.

Line 295 (Waxing lunar phases): For these experiments, which nest are you using? 1 or 2?

We have added that this is nest 1. 

Figure 3B: The title of this panel should be "Waxing Crescent Moon" I think. 

Ah yes, this is incorrect in the original submission. I have fixed this.

Lines 312-313: Here it sounds as though the ants went right back to the full +/- 45 degrees orientations when they clearly didn't (it was -26.6 degrees and 189.9 degrees). Maybe tone the language down a bit here.

Changed this to make clear the orientation shift is only ‘towards’ the ambient lunar e-vector.

Line 327: Insert "see" before "Figure 5" 

Added

Line 329: See comment for Line 295. 

We have added that this is nest 1. 

Lines 357-373 (Vector testing): Again, because of the somewhat confusing methods section describing these experiments, these results were hard to follow, both here and in the Discussion. I don't really understand what you have shown here. Re-think how you present this (and maybe re-working the Methods will be half the battle won). 

I have rewritten these sections to try to make clear these are ant tested with differences in vector length 6m vs. 2m, tested at the same location. Hopefully this is much clearer, but I think if these portions remain a bit confusing that a full rename of the conditions is in order. Something like long vector and short vector would help but comes with the problem of not truly describing what the purpose of the test is which is to control for location, thus the current condition names. As it stands, I hope the new clarifications adequately describe the reasoning while keeping the condition names. Of course, I am happy to make more changes here as making this clear to readers is important for driving home that the path integrator is in play.

See current change to results as an example: ‘Both forgers with a long ~6m remaining vector (Halfway Release), or a short ~2m remaining vector (Halfway Collection & Release), tested at the same location_,_ exhibited significant shifts to the right of initial headings when the e-vector was rotated clockwise +45°.’

Line 361: I think this should be 16.8 not 6.8 

Yes, you are correct. Fixed in text (16.8).

Line 365: I think this should be -12.7 not 12.7 

Yes, you are correct. Fixed in text (–12.7).

Line 408: "morning twilight". Should this be "morning solar twilight"? Plus "M midas" should be "M. midas"

Added and fixed respectively.

Line 440. "location" is spelt wrong. 

Fixed spelling.

Line 444: "...WITH longer accumulated vectors, ..." 

Added ‘with’ to sentence. 

Line 447: Remove "that just as"

Removed.

Line 448: "Moonlight polarised light" should be "Polarised moonlight" 

Corrected.

Lines 450-453: This sentence makes little sense scientifically or grammatically. A "limiting factor" can't be "accomplished". Please rephrase and explain in more detail.

This sentence has been rephrased:

‘The limiting factors to lunar cue use for navigation would instead be the ant’s detection threshold to either absolute light intensity, polarization sensitivity and spectral sensitivity. Moonlight is less UV rich compared to direct sunlight and the spectrum changes across the lunar cycle (Palmer and Johnsen 2015).’

Line 474: Re-write as "... due to the incorporation of the celestial compass into the path integrator..."

Added.

Reviewer #3 (Recommendations For The Authors): 

Minor comments 

Line 84 I am not sure that we can infer attentional processes in orientation to lunar skylight, at least it has not yet been investigated.

Yes, this is a good point. We have changed ‘attend’ to ‘use’.  

Line 90 This description of polarized light is a little vague; what is meant by the phrase "waves which occur along a single plane"? (What about the magnetic component? These waves can be redirected, are they then still polarized? Circular polarization?). I would recommend looking at how polarized light is described in textbooks on optics.

Response: We have rewritten the polarised light section to be clearer using optics and light physics for background. 

Line 92 The phrase "e-vector" has not been described or introduced up to this point.

We now introduce e-vector and define it. 

‘Polarised light comprises light waves which occur along a single plane and are produced as a by-product of light passing through the upper atmosphere (Horváth & Varjú 2004; Horváth et al., 2014). The scattering of this light creates an e-vector pattern in the sky, which is arranged in concentric circles around the sun or moon's position with the maximum degree of polarisation located 90° from the source. Hence when the sun/moon is near the horizon, the pattern of polarised skylight is particularly simple with uniform direction of polarisation approximately parallel to the north-south axes (Dacke et al., 1999, 2003; Reid et al. 2011; Zeil et al., 2014).’

Happy to make further changes as well.  

Line 107 Diurnal dung beetles can also orient to lunar skylight if roused at night (Smolka et al., 2016), provided the sky is bright enough. Perhaps diurnal ants might do the same?

Added the diurnal dung beetles mention as well as the reference.

Also, a very good suggestion using diurnal bull ants.

Line 146 Instead of lunar calendar the authors appear to mean "lunar cycle". 

Changed

Line 165 In Figure 1B, it looks like visual access to the sky was only partly "unobstructed". Indeed foliage covers as least part of the sky right up to the zenith.

We have added that the sky is partially obstructed. 

Line 179 This could also presumably be checked with a camera? 

For this testing we tried to keep equipment to a minimum for a single researcher walking to and from the field site given the lack of public transport between 1 and 4am. But yes, for future work a camera based confirmation system would be easier. 

Line 243 The abbreviation "PI" has not been described or introduced up to this point.

Changes to ‘path integration derived vector lengths….’

Line 267 The method for comparing the leftwards and rightwards shifts should be described in full here (presumably one set of shifts was mirrored onto the other?).

We have added the below description to indicate the full description of the mirroring done to counterclockwise shifts.

‘To assess shift magnitude between −45° and +45° foragers within conditions, we calculated the mirror of shift in each −45° condition, allowing shift magnitude comparisons within each condition. Mirroring the −45° conditions was calculated by mirroring each shift across the 0° to 180° plane and was then compared to the corresponding unaltered +45 condition.’

Discussion Might the brightness and spectrum of lunar skylight also play a role here?

We have added a section to the discussion to mention the aspects of moonlight which may be important to these animals, including the spectrum, brightness and polarisation intensity.  

Line 451 The sensitivity threshold to absolute light intensity would not be the only limiting factor here. Polarization sensitivity and spectral sensitivity may also play a role (moonlight is less UV rich than sunlight and the spectrum of twilight changes across the lunar cycle: Palmer & Johnsen, 2015). 

Added this clarification.

Line 478 Instead of the "masculine ordinal" symbol used (U+006F) here a degree symbol (U+00B0) should be used.

Ah thank you, we have replaced this everywhere in the text.  

Line 485 It should be possible to calculate the misalignment between polarization pattern before and after this interruption of celestial cues. Does the magnitude of this misalignment help predict the size of the reorientation?

Reorientations are highly correlated with the shift size under the filter, which makes sense as larger shifts mean that foragers need to turn back more to reorient to both the ambient pattern and to return to their visual route. Reorientation sizes do not show a consistent reduction compared to under-the-filter shifts when the lunar phase is low and is potentially harder to detect.

I have reworked this line in the text as I do not think there is much evidence for misalignment and it might be more precise to say that overnight periods where the moon is not visible may adversely impact the path integrator estimate, though it is currently unknown the full impact of this celestial cue gap of if other cues might also play a role.

Line 642 "from their" should be "relative to" 

Changed as requested

Figure 1B Some mention should be made of the differences in vegetation density. 

Added a sentence to the figure caption discussing the differences in both vegetation along the horizon and canopy cover.

Figures 2-6 A reference line at 0 degrees change might help the reader to assess the size of orientation changes visually. Confidence intervals around the mean orientation change would also help here.

We have now added circular grid lines and confidence intervals to the circular plots. These should help make the heading changes clear to readers.

This document was published 3 years after colony of Carolina was established. The Carolina colony was established March 24, 1963.