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eLife Assessment

This study presents useful findings on the role of the small GTPase Rab3A in homeostatic synaptic plasticity following activity suppression. While the study demonstrates that Rab3A is required for homeostatic scaling, the evidence supporting the model put forward by the authors is incomplete. The work will be of interest to the field of synaptic transmission and synaptic plasticity.

Reviewer #1 (Public review):

Koesters and colleagues investigated the role of the small GTPase Rab3A in homeostatic scaling of miniature synaptic transmission in primary mouse cortical cultures using electrophysiology and immunohistochemistry. The major finding is that TTX incubation for 48 hours does not induce an increase in the amplitude of excitatory synaptic miniature events in neuronal cultures derived from Rab3A KO and Rab3A Earlybird mutant mice. NASPM application had comparable effects on mEPSC amplitude in control and after TTX, implying that Ca2+-permeable glutamate receptors are unlikely modulated during synaptic scaling. Immunohistochemical analysis revealed no significant changes in GluA2 puncta size, intensity, and integral in control and Rab3A KO cultures. Finally, they provide evidence that loss of Rab3A in neurons, but not astrocytes, blocks homeostatic scaling. Based on these data, the authors propose a model in which neuronal Rab3A is required for homeostatic scaling of synaptic transmission through GluA2-dependent and independent mechanisms.

While the title of the manuscript is mostly supported by data of solid quality, many conclusions, as well as the final model, cannot be derived from the results presented. Importantly, the data do not support that GluA2 levels change upon TTX treatment in control cultures, rendering conclusions regarding Rab3A's role in TTX-dependent GluA2 modulation spurious. Other aspects of the model, such as a Rab3A-dependent release of a tropic factor, cannot be derived from the data.

The following points should be addressed:

(1) There is no (significant) increase in GluA2 levels (intensity, area, or integral) upon TTX treatment in controls (Fig. 5). Conclusions regarding Rab3As role in TTX-dependent GluA2 modulation should be revised accordingly. Hence, the data shown in Fig. 4 - 7 do not allow drawing conclusions in the context of Rab3A-dependent GluA2 modulation and scaling.

(2) The effects of Rab3A on TTX-induced mini frequency modulation remains unclear, because TTX does not induce a change in mini frequency in the Rab3A+/Ebd control (Fig. 2). The respective conclusions should be revised accordingly (l. 427).

(3) The model is still not supported by the data. In particular, data supporting a negative regulation of Rab3A by APs, Rab3A-dependent release of a tropic factor, or a Rab3A-dependent increase in GluA2 abundance are not presented.

(4) Data points are not overlapping and appear "quantal" in most box plots. How were the data rounded?

Reviewer #2 (Public review):

In the revised manuscript, the authors investigated the role of a presynaptic protein, Rab3A, in the homeostatic synaptic plasticity in cultured cortical neurons. The study was motivated by their previous findings that Rab3A is required for expression of similar homeostatic mechanisms at the neuromuscular junction. The authors first show that untreated WT neurons express homeostatic synaptic plasticity in response to 48h of TTX treatment (upregulation of both mEPSC amplitude and frequency), whereas neurons lacking Rab3A or carrying a dominant negative mutated Rab3A (earlybird) do not. They also demonstrate that only neuronal, but not glial Rab3A is responsible for this defect. Furthermore, they confirm the increased mEPSC amplitudes in WT neurons reflect the addition of GluA2-containing AMPA receptors rather than calcium-permeable ones, as previously reported by multiple labs. However, the increase in mEPSC amplitude is not accompanied by a corresponding upregulation of GluA2 synaptic clusters according to their IHC data (cluster size and intensity trend slightly upwards but not reaching significance). They further show that this modest upward trend is absent in Rab3A KO neurons, and conclude that Rab3A is involved in postsynaptic GluA2 upregulation during homeostatic synaptic plasticity.

When compared to the original version, the authors have done an admirable job in switching to more established ways to assess homeostatic synaptic plasticity by comparing the mean mEPSC amplitude and frequency, which has greatly improved the legibility of the manuscript to the public. Their data in Figures 1,2, and 8 clearly demonstrate that functional Rab3A in cortical neurons is required for the homeostatic regulation of mEPSCs.

However, the authors still have not provided further investigation of the mechanisms behind the role of Rab3A in this form of plasticity, and the revision therefore has added little to the significance of the study. Moreover, the experimental design for the investigation of the mismatch between mEPSC amplitude and GluA2 cluster fluorescence remains questionable, making it difficult to draw any credible conclusions from groups of data that not only look similar to the eye but also show no significance statistically.

A major claim the authors want to make is that Rab3A, although a presynaptic protein, regulates postsynaptic GluA2, and they do this by showing in Figure 5 that the upward trend of GluA2 cluster size and intensity is absent in Rab3A KO neurons. First, it is difficult to convince readers that this upward trend is real in Figures 5B-D without getting more samples. Second, the authors pick GluA2 clusters on the primary dendrites, whereas mEPSC events come from a much larger synapse population (e.g., more distal), therefore it makes sense that these two forms of measurement do not show matching changes, and this caveat could be addressed by sampling more diverse dendritic locations. Without a convincing phenotype in WT neurons, the support for this claim is weak.

Another claim of the authors is that this mismatch between mEPSC amplitude and GluA2 cluster sizes with the same culture suggests there are other factors contributing to the mEPSC amplitude. They do this by comparing results from individual culture dissociations, which greatly suffer from undersampling (Figure 6). In particular, they point out that 2 out of 3 dissociations show "matching" upward trends in mEPSC and GluA2 cluster (figure 6A and 6B) while the third one shows opposite trends, and use this to support their claim. Anyone who has done culture preparation would know the high variability between dissociations, which is why culture data are always pooled for assessment of any population trend. Anything could have happened to this particular dissociation (culture #3, figure 6C), and drawing conclusion from this single incident does little to support this claim. At least, they should double the dissociation numbers, and their claim would be much more convincing if a similar phenomenon occurs again. Besides, as mentioned above, all these mismatching trends could just be due to sampling differences.

In summary, this study establishes that neuronal Rab3A plays a role in homeostatic synaptic plasticity, but so do a number of other molecules that have been implicated in homeostatic synaptic plasticity in the past two decades (only will grow with the new techniques such as RNAseq). Without going beyond this finding and demonstrating how exactly Rab3A participates in the induction and/or expression of this form of plasticity, or maybe the potential Rab3A-mediated functional and behavioral defects in vivo, the contribution of the current study to the field is limited. However, given the presynaptic location of Rab3A, this finding could serve as a starting point for researchers interested in pre-postsynaptic cross-talk during homeostatic plasticity in general.

Reviewer #3 (Public review):

This manuscript presents a number of interesting findings that have the potential to increase our understanding of the mechanism underlying homeostatic synaptic plasticity (HSP). The data broadly support that Rab3A plays a role in HSP, although the site and mechanism of action remain uncertain.

The authors clearly demonstrate the Rab3A plays a role in HSP at excitatory synapses, with substantially less plasticity occurring in the Rab3A KO neurons. There is also no apparent HSP in the Earlybird Rab3A mutation, although baseline synaptic strength seems already elevated. In this context, it is unclear if the plasticity is absent or just occluded by a ceiling effect due the synapses already being strengthened. Occlusion may also occur in the mixed cultures, with Rab3A missing from neurons but not astrocytes. The authors do appropriately discuss both options. There are also differences in genetic background between the Rab3A KO and Earlybird mutants that could also impact the results, which are also noted. The authors have solid data showing that Rab3A is unlikely to be active in astrocytes, Finally, they attempt to study the linkage between synaptic strength during HSP and AMPA receptor trafficking and conclude that trafficking may not be solely responsible for the changes in synaptic strength.

Strengths:

This work adds another player into the mechanisms underlying an important form of synaptic plasticity. The plasticity is likely only reduced, suggesting Rab3A is only partially required and perhaps multiple mechanisms contribute. The authors speculate about some possible novel mechanisms.

However, the conclusions on the partial dissociation of AMPAR trafficking and synaptic response are made from somewhat weaker data. On average, across 3 culture sets, they saw similar magnitude of change in mEPSC amplitude and GluA2 cluster area and integral, but the GluA2 data was not significant. This is likely due to the nature of the datasets. Their imaging method involves only assessing puncta pairs (GluA2/VGlut1) clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, with usually less than 20 synapses per neuron analyzed (as stated by the authors). The mEPSC recordings will be averaging across several hundred events, which likely represent a hundred or more synapses given reasonable expectations on release probability. It has been reported, in work from this lab as well as by direct monitoring of tagged AMPARs during HSP (Wang, et al., 2019), that individual synapses are quite variable in their response. So there will almost necessarily be higher variability in the imaging data due to the smaller number of synapses sampled. The overall trends, though, are in alignment with previous data implicating receptor trafficking as the mechanism for HSP. However, the authors go on to evaluate each of the individual cultures, where 2 show similar changes between the mEPSC data and GluA2 clusters, and 1 culture showing little/no change in GluA2 clusters. The n's are very low here, and none of the datasets are significant. They want to conclude for this culture, there was a change in mEPSC amplitude that was not accompanied by a change in GluA2 at synaptic sites. But these data are collected from different coverslips, and due to the low n's, the potential under-sampling of the GluA2 clusters, and neuron-to-neuron variability, it is very hard to distinguish if this apparent difference is a methodological issue rather than a biological one. Much stronger data would be necessary to conclude that additional factors beyond receptor trafficking are required for HSP.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a frequency effect that is unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. However the change in frequency seems to argue (as the authors do) that some synapses only have CP-AMPARs, while the rest of the synapses have few or none. Another possibility is that there are pre-synaptic NASPM-sensitive receptors that influence release probability. Further, the amplitude data show a strong trend towards smaller amplitude following NASPM treatment (Fig 3B). The p value for both control and TTX neurons was 0.08 - it is very difficult to argue that there is no effect. The decrease on average is larger in the TTX neurons, and some cells show a strong effect. It is possible there is some heterogeneity between neurons on whether GluA1/A2 heteromers or GluA1 homomers are added during HSP. This would impact the weakly supported conclusions about the GluA2 imaging vs mEPSC amplitude data.

Unaddressed issues that would greatly increase the impact of the paper:

(1) Is Rab3A acting pre-synaptically, post-synaptically or both? The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where it is acting (pre or post) would aid substantially in understanding its role. They could use sparse knock-down of Rab3A, or simply mix cultures from KO and WT mice (with appropriate tags/labels). The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. The more support for their suggestion of a pre-synaptic site of control, the better.

(2) Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs (ie the opposite of whatever is happening at excitatory synapses). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling; an effect only at excitatory synapses would argue for a more specific role just at these synapses.

Author response:

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

The detailed, thorough critique provided by the three reviewers is very much appreciated. We believe the manuscript is greatly improved by the changes we have made based on those reviews. The major changes are described below, followed by a point by point response.

Major Changes:

(1) We revised our model (old Fig. 10; new Fig. 9) to keep the explanation focused on the data shown in the current study. Specifically, references to GTP/GDP states of Rab3A and changes in the presynaptic quantum have been removed and the mechanisms depicted are confined to pre- or post-synaptic Rab3A participating in either controlling release of a trophic factor that regulates surface GluA2 receptors (pre- or postsynaptic) or directly affecting fusion of GluA2-receptor containing vesicles (postsynaptic).

(2) We replaced all cumulative density function plots and ratio plots, based on multiple quantile samples per cell, with box plots of cell means. This affects new Figures 1, 2, 3, 5, 6, 7 and 8. All references to “scaling,” “divergent scaling,” or “uniform scaling,” have been removed. New p values for comparison of means are provided above every box plot in Figures 1, 2, 3, 5, 6, 7 and 8. The number of cultures is provided in the figure legends.

(3) We have added frequency to Figures 1, 2 and 8. Frequency values overall are more variable, and the effect of activity blockade less robust, than for mEPSC amplitudes. We have added text indicating that the increase in frequency after activity blockade was significant in neurons from cultures prepared from WT in the Rab3A+/- colony but not cultures prepared from KO mice (Results, lines 143 to 147, new Fig. 1G. H). The TTX-induced increase in frequency was significant in the NASPM experiments before NASPM, but not after NASPM (Results, lines 231 to 233, new Fig. 3, also cultures from WT in Rab3A+/- colony). The homeostatic plasticity effect on frequency did not reach significance in WT on WT glia cultures or

WT on KO glia cultures, possibly due to the variability of frequency, combined with smaller sample sizes (Results, lines 400 to 403, new Fig. 8). In the cultures prepared from WT mice in the Rab3A+/Ebd colony, there was a trend towards higher frequency after TTX that did not reach statistical significance, and in cultures prepared from mutant mice, the p value was large, suggesting disruption of the effect, which appears to be due to an increase in frequency in untreated cultures, similar to the behavior of mEPSC amplitudes in neurons from mutant mice (Results, lines 161-167). In sum, the effect of activity on frequency requires Rab3A and Ca2+-permeable receptors, and is mimicked by the presence of the Rab3A Earlybird mutant. We have also added a discussion of these results (Discussion, lines 427-435). 

(4) In the revised manuscript we have added analysis of VGLUT1 levels for the same synaptic sites that we previously analyzed GluA2 levels, and these data are described in Results, lines 344 to 371, and appear in new Table 2. In contrast to previous studies, we did not find any evidence for an increase in VGLUT1 levels after activity blockade. We reviewed those studies to determine whether there might be differences in the experimental details that could explain the lack of effect we observed. In (De Gois et al., 2005), the authors measured mRNA and performed western blots to show increases in VGLUT1 after TTX treatment in older rat cortical cultures (DIV 19). The study performs immunofluorescence imaging of VGLUT1 but only after bicuculline treatment (it decreases), not after TTX treatment. In (Wilson et al.,

2005), the hippocampal cultures are treated with AP5, not TTX, and the VGLUT1 levels in immunofluorescence images are reported relative to synapsin I. That the type of activity blockade matters is illustrated by the failure of Wilson and colleagues to observe a consistent increase in VGLUT1/Synapsin ratio in cultures treated with AMPA receptor blockade (NBQX; supplementary information). These points have been added to the Discussion, lines 436 to 447.)

Reviewer #1:

(1) (model…is not supported by the data), (2) (The analysis of mEPSC data using quantile sampling…), (3) (…statistical analysis of CDFs suffers from n-inflation…), (4) (How does recording noise and the mEPSC amplitude threshold affect “divergent scaling?”) (5) (…justification for the line fits of the ratio data…), (7) (A comparison of p-values between conditions….) and (10) (Was VGLUT intensity altered in the stainings presented in the manuscript?)

The major changes we made, described above, address Reviewer #1’s points. The remaining points are addressed below.

(6) TTX application induces a significant increase in mEPSC amplitude in Rab3A-/- mice in two out of three data sets (Figs. 1 and 9). Hence, the major conclusion that Rab3A is required for homeostatic scaling is only partially supported by the data. 

The p values based on CDF comparisons were problematic, but the point we were making is that they were much larger for amplitudes measured in cultures prepared from Rab3A-/- mice (Fig. 1, p = 0.04) compared to those from cultures prepared from Rab3A+/+ mice (Fig. 1, p = 4.6 * 10-4). Now that we are comparing means, there are no significant TTX-induced effects on mEPSC amplitudes for Rab3A-/- data. However, acknowledging that some increase after activity blockade remains, we describe homeostatic plasticity as being impaired or not significant, rather than abolished, by loss of Rab3A, (Abstract, lines 37 to 39; Results, lines 141 to 143; Discussion, lines 415 to 418).

(8) There is a significant increase in baseline mEPSC amplitude in Rab3AEbd/Ebd (15 pA) vs. Rab3AEbd/+ (11 pA) cultures, but not in Rab3A-/- (13.6 pA) vs. Rab3A+/- (13.9 pA). Although the nature of scaling was different between Rab3AEbd/Ebd vs. Rab3AEbd/+ and Rab3AEbd/Ebd with vs. without TTX, the question arises whether the increase in mEPSC amplitude in Rab3AEbd/Ebd is Rab3A dependent. Could a Rab3A independent mechanism occlude scaling?

The Reviewer is concerned that the increase in mEPSC amplitude in the presence of the Rab3A point mutant may be through a ‘non-Rab3A’ mechanism (a concern raised by the lack of such effect in cultures from the Rab3A-/- mice), and secondly, that the already large mEPSC cannot be further increased by the homeostatic plasticity mechanism. It must always be considered that a mutant with an altered genetic sequence may bind to novel partners, causing activities that would not be either facilitated or inhibited by the original molecule. We have added this caveat to Results, lines 180 to 186 We added that a number of other manipulations, implicating individual molecules in the homeostatic mechanism, have caused an increase in mEPSC amplitude at baseline, potentially nonspecifically occluding the ability of activity blockade to induce a further increase (Results lines 186 to 189). Still, it is a strong coincidence that the novel activity of the mutant Rab3A would affect mEPSC amplitude, the same characteristic that is affected by activity blockade in a Rab3A dependent manner, a point which we added to Results, lines 189 to 191.

(9) Figure 4: NASPM appears to have a stronger effect on mEPSC frequency in the TTX condition vs. control (-40% vs -15%). A larger sample size might be necessary to draw definitive conclusions on the contribution of Ca2+-permeable AMPARs.

Our results, even with the modest sample size of 11 cells, are clear: NASPM does not disrupt the effect of TTX treatment on mEPSC amplitude (new Fig. 3A). It also looks like there is a greater magnitude effect of NAPSM on frequency in TTX-treated cells; we note this, but point out that nevertheless, these mEPSCs are not contributing to the increase in mEPSC amplitude (Results, lines 238-241). 

(11) The change in GluA2 area or fluorescence intensity upon TTX treatment in controls is modest. How does the GluA2 integral change?

We had reported that GluA2 area showed the most prominent increase following activity blockade, with intensity changing very little. When we examined the integral, it closely matched the change in area. We have added the values for integral to new Fig. 5 D, H; new Fig. 6 A-C; new Fig. 7 A-C and new Table 1 (for GluA2) and new Table 2 (for VGLUT1). These results are described in the text in the following places: Results, lines 289-292; 298-299; 311-319; 328-324). For VGLUT1, both area and intensity changed modestly, and the integral appeared to be a combination of the two, being higher in magnitude and resulting in smaller p values than either area or intensity (Results, lines 344-348; 353-359; new Table 2).

(12) The quantitative comparison between physiology and microscopy data is problematic. The authors report a mismatch in ratio values between the smallest mEPSC amplitudes and the smallest GluA2 receptor cluster sizes (l. 464; Figure 8). Is this comparison affected by the fluorescence intensity threshold? What was the rationale for a threshold of 400 a.u. or 450 a.u.? How does this threshold compare to the mEPSC threshold of 3 pA.

This concern is partially addressed by no longer comparing the rank ordered mEPSC amplitudes with the rank ordered GluA2 receptor characteristics. We had used multiple thresholds in the event that an experiment was not analyzable with the chosen threshold (this in fact happened for VGLUT1, see end of this paragraph). We created box plots of the mean GluA2 receptor cluster size, intensity and integral, for experiments in which we used all three thresholds, to determine if the effect of activity blockade was different depending on which threshold was applied, and found that there was no obvious difference in the results (Author response image 1). Nevertheless, since there is no need to use a different threshold for any of the 6 experiments (3 WT and 3KO), for new Figures 5, 6 and 7 we used the same threshold for all data, 450; described in Methods, lines 746 to 749. For VGLUT1 levels, it was necessary to use a different threshold for Rab3A+/+ Culture #1 (400), but a threshold of 200 for the other five experiments (Methods, lines 751-757). The VGLUT1 immunofluorescent sites in Culture #1 had higher levels overall, and the low threshold caused the entire AOI to be counted as the synapse, which clearly included background levels outside of the synaptic site. Conversely, to use a threshold of 400 on the other experiments meant that the synaptic site found by the automated measurement tool was much smaller that what was visible by eye. In our judgement it would have been meaningless to adhere to a single threshold for VGLUT1 data.

Author response image 1.

Using different thresholds does not substantially alter GluA2 receptor cluster size data. A) Rab3A+/+ Culture #1, size data for three different thresholds, depicted above each graph. B) Rab3A+/+ Culture #2, size data for three different thresholds, depicted above each graph. Note scale bar in A is different from B, to highlight differences for different thresholds. (Culture #3 was only analyzed with 450 threshold).

The conclusion that an increase in AMPAR levels is not fully responsible for the observed mEPSC increase is mainly based on the rank-order analysis of GluA2 intensity, yielding a slope of ~0.9. There are several points to consider here: (i) GluA2 fluorescence intensity did increase on average, as did GluA2 cluster size.

(ii) The increase in GluA2 cluster size is very similar to the increase in mEPSC amplitude (each approx. 1820%). (iii) Are there any reports that fluorescence intensity values are linearly reporting mEPSC amplitudes (in this system)? Antibody labelling efficiency, and false negatives of mEPSC recordings may influence the results. The latter was already noted by the authors.

Our comparison between mEPSC amplitude and GluA2 receptor cluster characteristics has been reexamined in the revised version using means rather than rank-ordered data in rank-order plots or ratio plots. Importantly, all of these methods revealed that in one out of three WT cultures (Culture #3) GluA2 receptor cluster size (old Fig. 8, old Table 1; new Fig. 6, new Table 1), intensity and integral (new Fig. 6, new Table 1) values decreased following activity blockade while in the same culture, mEPSC amplitudes increased. It is based on this lack of correspondence that we conclude that increases in mEPSC amplitude are not fully explained by increases in GluA2 receptors, and suggest there may be other contributors. These points are made in the Abstract (lines 108-110); Results (lines 319 to 326; 330337; 341-343) and the Discussion (lines 472 to 474). To our knowledge, there are not any reports that quantitatively compare receptor levels (area, intensity or integrals) to mEPSC amplitudes in the same cultures. We examined the comparisons very closely for 5 studies that used TTX to block activity and examined receptor levels using confocal imaging at identified synapses (Hou et al., 2008; Ibata et al., 2008; Jakawich et al., 2010a; Xu and Pozzo-Miller, 2017; Dubes et al., 2022). We were specifically looking for whether the receptor data were more variable than the mEPSC amplitude data, as we found. However, for 4 of the studies, sample sizes were very different so that we cannot simply compare the p values. Below is a table of the comparisons.

Author response table 1.

In Xu 2017 the sample sizes are close enough that we feel comfortable concluding that the receptor data were slightly more variable (p < 0.05) than mEPSC data (p<0.01) but recognize that it is speculative to say our finding has been confirmed. A discussion of these articles is in Discussion, lines 456-474.

(iv) It is not entirely clear if their imaging experiments will sample from all synapses. Other AMPAR subtypes than GluA2 could contribute, as could kainite or NMDA receptors.

While our imaging data only examined GluA2, we used the application of NASPM to demonstrate Ca2+permeable receptors did not contribute quantitatively to the increase in mEPSC amplitude following TTX treatment. Since GluA3 and GluA4 are also Ca2+-permeable, the findings in new Figure 3 (old Fig. 4) likely rule out these receptors as well.  There are also reports that Kainate receptors are Ca2+-permeable and blocked by NASPM (Koike et al., 1997; Sun et al., 2009), suggesting the NASPM experiment also rules out the contribution of Kainate receptors. Finally, given our recording conditions, which included normal magnesium levels in the extracellular solution as well as TTX to block action-potential evoked synaptic transmission, NMDA receptors would not be available to contribute currents to our recordings due to block by magnesium ions at resting Vm. These points have been added to the Methods section, lines 617 to 677 (NMDA); 687-694 (Ca2+-permeable AMPA receptors and Kainate receptors).

Furthermore, the statement “complete lack of correspondence of TTX/CON ratios” is not supported by the data presented (l. 515ff). First, under the assumption that no scaling occurs in Rab3A-/-, the TTX/CON ratios show a 20-30% change, which indicates the variation of this readout. Second, the two examples shown in Figure 8 for Rab3A+/+ are actually quite similar (culture #1 and #2, particularly when ignoring the leftmost section of the data, which is heavily affected by the raw values approaching zero.

We are no longer presenting ratio plots in the revised manuscript, so we do not base our conclusion that mEPSC amplitude data is not always corresponding to GluA2 receptor data on the difference in behavior of TTX/CON ratio values, but only on the difference in direction of the TTX effect in one out of three cultures. We agree with the reviewer that the ratio plots are much more sensitive to differences between control and treated values than the rank order plot, and we feel these differences are important, for example, there is still a homeostatic increase in the Rab3A-/- cultures, and the effect is still divergent rather than uniform. But the comparison of ratio data will be presented elsewhere.

(13) Figure 7A: TTX CDF was shifted to smaller mEPSC amplitude values in Rab3A-/- cultures. How can this be explained?

While this result is most obvious in CDF plots, we still observe a trend towards smaller mEPSC amplitudes after TTX treatment in two of three individual cultures prepared from Rab3A-/- mice when comparing means (new Fig. 7, Table 1) which did not reach statistical significance for the pooled data (new Fig. 5, new Table 1). There was not any evidence of this decrease in the larger data set (new Fig. 1) nor for Rab3A-/- neurons on Rab3A+/+ glia (new Fig. 8). Given that this effect is not consistent, we did not comment on it in the revised manuscript. It may be that there is a non-Rab3A-dependent mechanism that results in a decrease in mEPSC amplitude after activity blockade, which normally pulls down the magnitude of the activity-dependent increase typically observed. But studying this second component would be difficult given its magnitude and inconsistent presentation.

Reviewer #1 (Recommendations For the Authors):

(1) Abstract, last sentence: The conclusion of the present manuscript should be primarily based on the results presented. At present, it is mainly based on a previous publication by the authors.

We have revised the last sentence to reflect actual findings of the current study (Abstract, lines 47 to 49).

(2) Line 55: “neurodevelopmental”

This phrase has been removed.

(3) Line 56: “AMPAergic” should be replaced by AMPAR-mediated

This sentence was removed when all references to “scaling” were removed; no other instances of “AMPAergic” are present.

(4) Figure 9: The use of BioRender should be disclosed in the Figure Legend.

We used BioRender in new Figures 3, 7 and 8, and now acknowledge BioRender in those figure legends.

(5) Figure legends and results: The number of cultures should be indicated for each comparison.

Number of cultures has been added to the figure legends.

(6) Line 289: A comparison of p-values between conditions does not allow any meaningful conclusions.

Agreed, therefore we have removed CDFs and the KS test comparison p values. All comparisons in the revised manuscript are for cell means.

(7) Line 623ff: The argument referring to NMJ data is weak, given that different types of receptors are involved.

We still think it is valid to point out that Rab3A is required for the increase in mEPC at the NMJ but that ACh receptors do not increase (Discussion, lines 522 to 525). We are not saying that postsynaptic receptors do not contribute in cortical cultures, only that there could be another Rab3A-dependent mechanism that also affects mEPSC amplitude.

(8) Plotting data points outside of the ranges should be avoided (e.g., Fig. 2Giii, 7F).

These two figures are no longer present in the revised manuscript. In revising figures, we made sure no other plots have data points outside of the ranges.

(9) The rationale for investigating Rab3AEbd/Ebd remains elusive and should be described.

A rationale for investigating Rab3AEbd/Ebd is that if the results are similar to the KO, it strengthens the evidence for Rab3A being involved in homeostatic synaptic plasticity. In addition, since its phenotype of early awakening was stronger than that demonstrated in Rab3A KO mice (Kapfhamer et al., 2002), it was possible we would see a more robust effect. These points have been added to the Results, lines 118 to 126.

(10) Figures 3 and 4, as well as Figure 5 and 6 could be merged.

In the revised version, Figure 3 has been eliminated since its main point was a difference in scaling behavior. Figure 4 has been expanded to include a model of how NASPM could reduce frequency (new Fig. 3.) Images of the pyramidal cell body have been added to Figure 5 (new Fig. 4), and Figure 6 has been completely revised and now includes pooled data for both Rab3A+/+ and Rab3A-/- cultures, for mEPSC amplitude, GluA2 receptor cluster size, intensity and integral.

(11) Figure 5: The legend refers to MAP2, but this is not indicated in the figure.

MAP2 has now been added to the labels for each image and described in the figure legend (new Fig. 4).

Reviewer #2:

Technical concerns:

(1) The culture condition is questionable. The authors saw no NMDAR current present during spontaneous recordings, which is worrisome since NMDARs should be active in cultures with normal network activity (Watt et al., 2000; Sutton et al., 2006). It is important to ensure there is enough spiking activity before doing any activity manipulation. Similarly it is also unknown whether spiking activity is normal in Rab3AKO/Ebd neurons.

In the studies cited by the reviewer, NMDA currents were detected under experimental conditions in which magnesium was removed. In our recordings, we have normal magnesium (1.3 mM) and also TTX, which prevents the necessary depolarization to allow inward current through NMDA receptors. This point has been added to our Methods, lines 674 to 677. We acknowledge we do not know the level of spiking in cultures prepared from Rab3A+/+, Rab3A-/- or Rab3A_Ebd/Ebd_ mice. Given the similar mEPSC amplitude for untreated cultures from WT and KO studies, we think it unlikely that activity was low in the latter, but it remains a possibility for untreated cultures from Rab3A_Ebd/Ebd_ mice, where mEPSC amplitude was increased. These points are added to the Methods, lines 615 to 622.

(2) Selection of mEPSC events is not conducted in an unbiased manner. Manually selecting events is insufficient for cumulative distribution analysis, where small biases could skew the entire distribution. Since the authors claim their ratio plot is a better method to detect the uniformity of scaling than the well-established rank-order plot, it is important to use an unbiased population to substantiate this claim.

We no longer include any cumulative distributions or ratio plot analysis in the revised version. We have added the following text to Methods, lines 703 to 720:

“MiniAnalysis selects many false positives with the automated feature when a small threshold amplitude value is employed, due to random fluctuations in noise, so manual re-evaluation of the automated process is necessary to eliminate false positives. If the threshold value is set high, there are few false positives but small amplitude events that visually are clearly mEPSCs are missed, and manual re-evaluation is necessary to add back false negatives or the population ends up biased towards large mEPSC amplitudes. As soon as there is a manual step, bias is introduced. Interestingly, a manual reevaluation step was applied in a recent study that describes their process as ‘unbiased (Wu et al., 2020). In sum, we do not believe it is currently possible to perform a completely unbiased detection process. A fully manual detection process means that the same criterion (“does this look like an mEPSC?”) is applied to all events, not just the false positives, or the false negatives, which prevents the bias from being primarily at one end or the other of the range of mEPSC amplitudes. It is important to note that when performing the MiniAnalysis process, the researcher did not know whether a record was from an untreated cell or a TTX-treated cell.”

(3) Immunohistochemistry data analysis is problematic. The authors only labeled dendrites without doing cell-fills to look at morphology, so it is questionable how they differentiate branches from pyramidal neurons and interneurons. Since glutamatergic synapse on these two types of neuron scale in the opposite directions, it is crucial to show that only pyramidal neurons are included for analysis.

We identified neurons with a pyramidal shape and a prominent primary dendrite at 60x magnification without the zoom feature. This should have been made clear in the description of imaging. We have added an image of the two selected cells to our figure of dendrites (old Fig. 5, new Fig. 4), and described this process in the Methods, lines 736 to 739, and Results, lines 246 to 253. Given the morphology of the neurons selected it is highly unlikely that the dendrites we analyzed came from interneurons.

Conceptual Concerns

The only novel finding here is the implicated role for Rab3A in synaptic scaling, but insights into mechanisms behind this observation are lacking. The authors claim that Rab3A likely regulates scaling from the presynaptic side, yet there is no direct evidence from data presented. In its current form, this study’s contribution to the field is very limited.

We have demonstrated that loss of Rab3A and expression of a Rab3A point mutant disrupt homeostatic plasticity of mEPSC amplitudes, and that in the absence of Rab3A, the increase in GluA2 receptors at synaptic sites is abolished. Further, we show that this effect cannot be through release of a factor, like TNFα, from astrocytes. In the new version, we add the finding that VGLUT1 is not increased after activity blockade, ruling out this presynaptic factor as a contributor to homeostatic increases in mEPSC amplitude. We show for the first time by examining mEPSC amplitudes and GluA2 receptors in the same cultures that the increases in GluA2 receptors are not as consistent as the increases in mEPSC amplitude, suggesting the possibility of another contributor to homeostatic increases in mEPSC amplitude. We first proposed this idea in our previous study of Rab3A-dependent homeostatic increases in mEPC amplitudes at the mouse neuromuscular junction. In sum, we dispute that there is only one novel finding and that we have no insights into mechanism. We acknowledge that we have no direct evidence for regulation from the presynaptic side, and have removed this claim from the revised manuscript. We have retained the Discussion of potential mechanisms affecting the presynaptic quantum and evidence that Rab3A is implicated in these mechanisms (vesicle size, fusion pore kinetics; Discussion, lines 537 to 563). One way to directly show that the amount of transmitter released for an mEPSC has been modified after activity blockade is to demonstrate that a fast off-rate antagonist has become less effective at inhibiting mEPSCs (because the increased glutamate released out competes it; see (Liu et al., 1999) and (Wilson et al., 2005) for example experiments). This set of experiments is underway but will take more time than originally expected, because we are finding surprisingly large decreases in frequency, possibly the result of mEPSCs with very low glutamate concentration that are completely inhibited by the dose used. Once mEPSCs are lost, it is difficult to compare the mEPSC amplitude before and after application of the antagonist. Therefore we intend to include this experiment in a future report, once we determine the reason for the frequency reduction, or, can find a dose where this does not occur.

(1) Their major argument for this is that homeostatic effects on mEPSC amplitudes and GluA2 cluster sizes do not match. This is inconsistent with reports from multiple labs showing that upscaling of mEPSC amplitude and GluA2 accumulation occur side by side during scaling (Ibata et al., 2008; Pozo et al., 2012; Tan et al., 2015; Silva et al., 2019). Further, because the acquisition and quantification methods for mEPSC recordings and immunohistochemistry imaging are entirely different (each with its own limitations in signal detection), it is not convincing that the lack of proportional changes must signify a presynaptic component.

Within the analyses in the revised manuscript, which are now based only on comparison of cell/dendrite means, we find a very good match in the magnitude of increase for the pooled data of mEPSC amplitudes and GluA2 receptor cluster sizes (+19.7% and +20.0% respectively; new Table 1). However, when looking at individual cultures, we had one of three WT cultures in which mEPSC amplitude increased 17.2% but GluA2 cluster size decreased 9.5%. This result suggests that while activity blockade does lead to an increase in GluA2 receptors after activity blockade, the effect is more variable than that for mEPSC amplitude. We went back to published studies to see if this has been previously observed, but found that it was difficult to compare because the sample sizes were different for the two characteristics (see Author response table 1). We included these particular 5 studies because they use the same treatment (TTX), examine receptors using imaging of identified synaptic sites, and record mEPSCs in their cultures (although the authors do not indicate that imaging and recordings are done simultaneously on the same cultures.) Only one of the studies listed by the Reviewer is in our group (Ibata et al., 2008). The study by (Tan et al., 2015) uses western blots to measure receptors; the study by (Silva et al., 2019) blocks activity using a combination of AMPA and NMDA receptor blockers; the study by (Pozo et al., 2012) correlates mEPSC amplitude changes with imaging but not in response to activity blockade, instead for changing the expression of GluA2. While it may seem like splitting hairs to reject studies that use other treatment protocols, there is ample evidence that the mechanisms of homeostatic plasticity depend on how activity was altered, see the following studies for several examples of this (Sutton et al., 2006; Soden and Chen, 2010; Fong et al., 2015). A discussion of the 5 articles we selected is in the revised manuscript, Discussion, lines 456 to 474. In sum, we provide evidence that activity blockade is associated with an overall increase in GluA2 receptors; what we propose is that this increase, being more variable, does not fully explain the increase in mEPSC amplitude. However, we acknowledge that the disparity could be explained by the differences in limitations of the two methods (Discussion, lines 469-472).

(2) The authors also speculate in the discussion that presynaptic Rab3A could be interacting with retrograde BDNF signaling to regulate postsynaptic AMPARs. Without data showing Rab3A-dependent presynaptic changes after TTX treatment, this argument is not compelling. In this retrograde pathway, BDNF is synthesized in and released from dendrites (Jakawich et al., 2010b; Thapliyal et al., 2022), and it is entirely possible for postsynaptic Rab3A to interfere with this process cell-autonomously.

We have added the information that Rab3A could control BDNF from the postsynaptic cell and included the two references provided by the reviewer, Discussion, lines 517 to 518. We have added new evidence, recently published, that the Rab3 family has been shown to regulate targeting of EGF receptors to rafts (among other plasma membrane molecules), with Rab3A itself clearly present in nonneuronal cells (Diaz-Rohrer et al., 2023) (added to Discussion, lines 509 to 515).

(3) The authors propose that a change in AMPAR subunit composition from GluA2-containing ones to GluA1 homomers may account for the distinct changes in mEPSC amplitudes and GluA2 clusters. However, their data from the NASPM wash-in experiments clearly show that the GluA1 homomer contributions have not changed before and after TTX treatment.

We have revised this section in the Discussion, lines 534 to 536, to clarify that any change due to GluA1 homomers should have been detectable by a greater ability of NASPM to reverse the TTX-induced increase.

Reviewer #2 (Recommendations for the Authors):

For authors to have more convincing arguments in general, they will need to clarify/improve certain details in their data collection by addressing the above technical concerns. Additionally, the authors should design experiments to test whether Rab3A regulates scaling from pre- or post-synaptic site. For example, they could sparsely knock out Rab3A in WT neurons to test the postsynaptic possibility. On the other hand, their argument for a presynaptic role would be much more compelling if they could show whether there are clear functional changes such as in vesicle sizes and release probability in the presynaptic terminal of Rab3AKO neurons.

An important next step is to identify whether Rab3A is acting pre- or post-synaptically (Discussion, lines 572 to 573), but these experiments will be undertaken in the future. It would not add much to simply show vesicle size is altered in the KO (and we do not necessarily expect this since mEPSC amplitude is normal in the KO). It will be very difficult to establish that vesicle size is changing with activity blockade and that this change is prevented in the Rab3A KO, because we are looking for a ~25% increase in vesicle volume, which would correspond to a ~7.5% increase in diameter. Finally, we do not believe demonstrating changes in release probability tell us anything about a presynaptic role for Rab3A in regulating the size of the presynaptic quantum.

Reviewer #3 (Public Review)

Weaknesses: However, the rather strong conclusions on the dissociation of AMPAR trafficking and synaptic response are made from somewhat weaker data. The key issue is the GluA2 immunostaining in comparison with the mEPSC recordings. Their imaging method involves only assessing puncta clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, judging from the sample micrographs (Fig. 5). To my knowledge, this is a new and unvalidated approach that could represent a particular subset of synapses not representative of the synapses contributing to the mEPSC change (they are also sampling different neurons for the two measurements; an additional unknown detail is how far from the cell body were the analyzed dendrites for immunostaining.) While the authors acknowledge that a sampling issue could explain the data, they still use this data to draw strong conclusions about the lack of AMPAR trafficking contribution to the mEPSC amplitude change. This apparent difference may be a methodological issue rather than a biological one, and at this point it is impossible to differentiate these. It will unfortunately be difficult to validate their approach. Perhaps if they were to drive NMDAdependent LTD or chemLTP, and show alignment of the imaging and ephys, that would help. More helpful would be recordings and imaging from the same neurons but this is challenging. Sampling from identified synapses would of course be ideal, perhaps from 2P uncaging combined with SEP-labeled AMPARs, but this is more challenging still. But without data to validate the method, it seems unwarranted to make such strong conclusions such as that AMPAR trafficking does not underlie the increase in mEPSC amplitude, given the previous data supporting such a model.

In the new version, we soften our conclusion regarding the mismatch between GluA2 receptor levels and mEPSC amplitudes, now only stating that receptors may not be the sole contributor to the TTX effect on mEPSC amplitude (Discussion, lines 472 to 474). With our analysis in the new version focusing on comparisons of cell means, the GluA2 receptor cluster size and the mEPSC amplitude data match well in magnitude for the data pooled across the 3 matched cultures (20.0% and 19.7%, respectively, see new Table 1). However, in one of the three cultures the direction of change for GluA2 receptors is opposite that of mEPSC amplitudes (Table 1, Culture #3, -9.5% vs +17.2%, respectively).

It is unlikely that the lack of matching of homeostatic plasticity in one culture, but very good matching in two other cultures, can be explained by an unvalidated focus on puncta associated with MAP2 positive dendrites. We chose to restrict analysis of synaptic GluA2 receptors to the primary dendrite in order to reduce variability, reasoning that we are always measuring synapses for an excitatory pyramidal neuron, synapses that are relatively close to the cell body, on the consistently identifiable primary dendrite. We measured how far this was for the two cells depicted in old Figure 5 (new Fig. 4). Because we always used the 5X zoom window which is a set length, and positioned it within ~10 microns of the cell body, these cells give a ball park estimate for the usual distances. For the untreated cell, the average distance from the cell body was 38.5 ± 2.8 µm; for the TTX-treated cell, it was 42.4 ± 3.2 µm (p = 0.35, KruskalWallis test). We have added these values to the Results, lines 270 to 274.

We did not mean to propose that AMPA receptor levels do not contribute at all to mEPSC amplitude, and we acknowledge there are clear cases where the two characteristics change in parallel (for example, in the study cited by Reviewer #2, (Pozo et al., 2012), increases in GluA2 receptors due to exogenous expression are closely matched by increases in mEPSC amplitudes.) What our matched culture experiments demonstrate is that in the case of TTX treatment, both GluA2 receptors and mEPSC amplitudes increase on average, but sometimes mEPSC amplitudes can increase in the absence of an increase in GluA2 receptors (Culture #3, Rab3A+/+ cultures), and sometimes mEPSC amplitudes do not increase even though GluA2 receptor levels do increase (Culture #3, Rab3A-/- cultures). Therefore, it would not add anything to our argument to examine receptors and mEPSCs in NMDA-dependent LTP, a different plasticity paradigm in which changes in receptors and mEPSCs may more closely align. It has been demonstrated that mEPSCs of widely varying amplitude can be recorded from a single synaptic site (Liu and Tsien, 1995), so we would need to measure a large sample of individual synapse recordings to detect a modest shift in average values due to activity blockade. In addition, it would be essential to express fluorescent AMPA receptors in order to correlate receptor levels in the same cells we record from (or at the same synapses). And yet, even after these heroics, one is still left with the issue that the two methods, electrophysiology and fluorescent imaging, have distinct limitations and sources of variability that may obscure any true quantitative correlation.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a frequency effect that is quite unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. It is also unclear why the authors argue this proves that NASPM was at an effective concentration (lines 399-400). Further, the amplitude data show a strong trend towards smaller amplitude. The p value for both control and TTX neurons was 0.08 – it is very difficult to argue that there is no effect. And the decrease is larger in the TTX neurons. Considering the strong claims for a presynaptic locus and the use of this data to justify only looking at GluA2 by immunostaining, these data do not offer much support of the conclusions. Between the sampling issues and perhaps looking at the wrong GluA subunit, it seems premature to argue that trafficking is not a contributor to the mEPSC amplitude change, especially given the substantial support for that hypothesis. Further, even if trafficking is not the major contributor, there could be shifts in conductance (perhaps due to regulation of auxiliary subunits) that does not necessitate a pre-synaptic locus. While the authors are free to hypothesize such a mechanism, it would be prudent to acknowledge other options and explanations.

We have created a model cartoon to explain how NASPM could reduce mEPSC frequency (new Fig. 3D). mEPSCs that arise from a synaptic site that has only Ca2+-permeable AMPA receptors will be completely blocked by NASPM, if the NASPM concentration is maximal. The reason we conclude that we have sufficient NASPM reaching the cells is that the frequency is decreased, as expected if there are synaptic sites with only Ca2+-permeable AMPA receptors. We previously were not clear that there is an effect of NASPM on mEPSC amplitude, although it did not reach statistical significance (new Fig. 3B). Where there is no effect is on the TTX-induced increase in mEPSC amplitude, which remains after the acute NASPM application (new Fig. 3A). We have revised the description of these findings in Results, lines 220 to 241. In reviewing the literature further, we could find no previous studies demonstrating an increase in conductance in GluA2 or Ca2+-impermeable receptors, only in GluA1 homomers. In other words, any conductance change would have been due to a change in GluA1 homomers, and should have been visible as a disruption of the homeostatic plasticity by NASPM application. We have added text to Results, lines 211 to 217; 236-241; Discussion, lines 420 to 422; 526-536 and Methods, lines 685 to 695 regarding this point.

The frequency data are missing from the paper, with the exception of the NASPM dataset. The mEPSC frequencies should be reported for all experiments, particularly given that Rab3A is generally viewed as a pre-synaptic protein regulating release. Also, in the NASPM experiments, the average frequency is much higher in the TTX treated cultures. Is this statistically above control values?

This comment is addressed by the major change #3, above.

Unaddressed issues that would greatly increase the impact of the paper:

(1) Is Rab3A activity pre-synaptically, post-synaptically or both. The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where is it acting (pre or post) would aid substantially in understanding its role (and particularly the hypothesized and somewhat novel idea that the amount of glutamate released per vesicle is altered in HSP). They could use sparse knockdown of Rab3A, or simply mix cultures from KO and WT mice (with appropriate tags/labels). The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. The more support for their suggestion of a pre-synaptic site of control, the better.

This is similar to the request of Reviewer #2, Recommendations to the Authors. An important next step is to identify whether Rab3A is working pre- or postsynaptically. However, it is possible that it is acting pre-synaptically to anterogradely regulate trafficking of AMPAR, as we have depicted in our model, new Fig. 9. To demonstrate that the presynaptic quantum is being altered, we would need to show that vesicle size is increased, or the amount of transmitter being released during an mEPSC is increased after activity blockade. To that end, we are currently performing experiments using a fast off-rate antagonist. As described above in response to Reviewer #2’s Conceptual Concerns, we find dramatic decreases in frequency not explained by the 30-60% inhibition observed for the largest amplitude mEPSCs, which suggests the possibility that small mEPSCs are more sensitive than large mEPSCs and therefore may have less transmitter. Due to these complexities and the delay while we test other antagonists to see if the effect is specific to fast-off rate antagonists, we are not including these results here.

(2) Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs and/or a decrease of GABA-packaging in vesicles (ie the opposite of whatever is happening at excitatory synapses.). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling, an effect only at excitatory synapses would argue for a more specific role just at these synapses.

It will be important to determine if homeostatic synaptic plasticity at inhibitory synapses on excitatory neurons is sensitive to Rab3A deletion, especially in light of the fact that unlike many of the other molecules implicated in homeostatic increases in mEPSCS, Rab3A is not a molecule known to be selective for glutamate receptor trafficking (in contrast to Arc/Arg3.1 or GRIP1, for example). Such a study would warrant its own publication.

Reviewer #3 (Recommendations for the Authors):

There are a number of minor points or suggestions for the authors:

Is RIM1 part of this pathway (or expected to be)? Some discussion of this would be nice.

RIM, Rab3-interacting molecule, has been implicated at the drosophila neuromuscular junction in a presynaptic form of homeostatic synaptic plasticity in which evoked release is increased after block of postsynaptic receptors (Muller et al., 2012), a plasticity that also requires Rab3-GAP (Muller et al., 2011). To our knowledge there is no evidence that RIM is involved in the homeostatic plasticity of mEPSC amplitude after activity blockade by TTX. The Rim1a KO does not have a change in mEPSC amplitude relative to WT (Calakos et al., 2004), but that is not unexpected given the normal mEPSC amplitude in neurons from cultures prepared from Rab3A-/- mice in the current study. It would be interesting to look at homeostatic plasticity in cortical cultures prepared from Rim1a or other RIM deletion mice, but we have not added these points to the revised manuscript since there are a number of directions one could go in attempting to define the molecular pathway and we feel it is more important to discuss the potential location of action and physiological mechanisms.

Is the Earlybird mutation a GOF? More information about this mutation would help.

We have added a description of how the Earlybird mutation was identified, in a screen for rest:activity mutants (Results, lines 118 to 123). Rab3A Earlybird mice have a shortened circadian period, shifting their wake cycle earlier and earlier. When Rab3A deletion mice were tested in the same activity raster plot measurements, the shift was smaller than that for the Earlybird mutant, suggesting the possibility that it is a dominant negative mutation.

The high K used in the NASPM experiments seems a bit unusual. Have the authors done high K/no drug controls to see if this affects the synapses in any way?

We used the high K based on previous studies that indicated the blocking effect of the Ca2+-permeable receptor blockers was use dependent (Herlitze et al., 1993; Iino et al., 1996; Koike et al., 1997). We reasoned that a modest depolarization would increase the frequency of AMPA receptor mEPSCs and allow access of the NASPM.  We have added this point to the Methods, lines 695 to 708. 

The NASPM experiments do not show that GluA1 does not contribute (line 401), only that GluA1 homomers are not contributing (much – see above). GluA1/A2 heteromers are quite likely involved. Also, the SEM is missing from the WT pre/post NASPM data.

Imaging of GluA2-positive sites will not distinguish between GluA2 homomers and GluA2-GluA1 heteromers, so we have added this clarification to Results, lines 242 to 246. We have remade the NASPM pre-post line plots so that the mean values and error bars are more visible (new Fig. 3B, C).

It seems odd to speculate based on non-significant findings (line 650-1), with lower significance (p = 0.11) than findings being dismissed in the paper (NASPM on mEPSC amplitude; p = 0.08).

We did not mean to dismiss the effect of NASPM on mEPSC amplitude (new Fig. 3B), rather, we dismiss the effect of NASPM on the homeostatic increase in mEPSC amplitude caused by TTX treatment (new Fig. 3A). We have emphasized this distinction in Results, lines 223 to 225, and Discussion, lines 420 to 422, as well as adding that the stronger effect of NASPM on frequency after TTX treatment suggests an activity-dependent increase in the number of synapses expressing only Ca2+ permeable homomers (Results, lines 236 to 241; Discussion, lines 431 to 435).

Fig. 4 could be labeled better (to make it clear that B is amplitude and C is freq from the same cells).

Fig. 4 has been revised—now the amplitude and frequency plots from the same condition (new Fig. 3, B, C; CON or TTX) are in a vertical line and the figure legend states that the frequency data are from the same cells as in Fig. 3A.

The raw amplitude data seems a bit hidden in the inset panels – I would suggest these data are at least as important as the cumulative distributions in the main panel. Maybe re-organizing the figures would help.

We have removed all cumulative distributions, rank order plots, and ratio plots. The box plots are now full size in new Figures 1, 2, 5, 6, 7 and 8.

I’m not sure I would argue in the paper that 12 cells a day is a limiting issue for experiments. It doesn’t add anything and doesn’t seem like that high a barrier. It is fine to just say it is difficult and therefore there is a limited amount of data meeting the criteria.

We have removed the comment regarding difficulty.

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Bold

for - question - @Gyuri - How would Indyweb do this?

Not only is this story funny but it did get me thinking: What do you do when somebody tries to sabotage your legal research process? Sure, such a process may be unethical and likely to result in those who sabotage their opponents being punished but that does not mean it is not done. If you not only have to deal with the difficulties of legal research but also of somebody trying to prevent you from doing legal research that may be a situation rare enough for few to write about yet risky enough to be concerning for those doing legal research.

Reading this initially this comment really stood out to me because of how odd it was. "What is he talking about? You can easily find a hundred different research outlines with a simple search." I looked up the paper and found out it was published in 1953. These sentences show how different the legal world has become in just 70 years. With the advent of the internet and websites such as Westlaw and Lexis the difficulty of doing legal research has substantially decreased. There was once a time where even the knowledge of how to research was scarce and I am glad that time no longer exists.

There is a lot of antisocial media information online. Part of those people who get involved in these steams this way could attract people's attention and make more people notice their idea. So the "rule" build by the social media platform is necessary, such as the limitation of specific bad words and high attention and sensibility to potential anti-social behaviors.

Good advice for the presenters using macOS

When we first get in touch with the name and content of this course and book. The limited thinking was social media only included platforms online. But we forgot about what those online stuff comes from and retrieve from. People's passion and curiosity in the public contribute to the development of society and future things.

Deliver Progressive Web Apps with the capability of allowing fast, reliable, and engaging user experiences across platforms. PWAs are the best of both worlds, taking all that a web app has to offer you and pairing it with everything that its rival-the mobile application-provides, thus including offline functionality, push notifications, and an interface similar to that of an app-but without the app store. Whether better engagement or performance, PWAs are a cost-effective option for business companies.

Got an error message when running this code. The code below worked for me:

stargazer(TSMA_mod1, TSMA_mod2, TSMA_mod3, TSMA_mod4, TSMA_mod5, TSMA_mod6, title = "Regressions Using Massachusetts Test Score Data", type = "text", digits = 3, se = rob_se, dep.var.caption = "Dependent Variable: Test Score", column.labels = c("(I)", "(II)", "(III)", "(IV)", "(V)", "(VI)"))

This last line shows that despite her efforts to obliterate herself and thus (end the novel), it is in vain, there is a hopeful light in her abduction!

I think this is a hopeful ending, as out of the many options she lays out for us on what she can do -- there is a new option, to be taken away. Not to be killed in a salvaging or hung on the wall, but just taken away-- She has opened an escape outside of herself.

Naming is such a powerful mark of identity. In it, it defers what the object/person actually is. The executions are thus called salvagings, the abusers are called Aunts as a form of a mother's harsh love, and the place of happiness is called Jezebel's. In a sense, morality is shifted (ironically) through the use of contradicting nomenclature.

The longest consideration -- resembling Moira, and a sense of power in her demise and death.

I feel like this is a very smart approach by the designers as sometimes the "x" is too small and can't be pressed so it just opens the website instead or it takes a while to show up, in most of the users are too lazy to go back to what they were doing and end up watching the whole ad. This for sure leads to the product getting views and even sales. It is so crazy how the whole world is hooked to social media and their phones all day and the designers just make money out of it

Knowing vs Witnessing, witnessing behavior like this and also going through it causes so much trauma. It shows how inhumane people treated them.

Passing down history through family has such a warm meaningful feeling. Knowing that this information comes from your ancestry must mean a lot.

Understanding what it means to be a Dakota woman, is such a powerful statement due to what she unravels later in the text.

I find it interesting how complications are simplified overtime if in the 1980s and 1990s, BBS was commonly used, to me it doesn't seem very intriguing as if it looks very dry without any pictures or a comment section where everyone can fight lol, like if we can not post memes on social media what's the point. However now, social media has become so much more simple and fun. We go to social media apps such as TikTok or Instagram to take our mind off from work and if I see the picture of BBS it looks like coding, so I wouldn't really use a site that reminds me of work even in my free time.

Shifting our focus will help us better understand techniques that would genuinely be useful for students. This will help them learn better and grasp the material easier.

The fact that was written in 2011, and people still feel like this in 2024 shows us how we haven't improved.

This is a point that many teachers should pay attention to, not everyone is good at test taking. There are other ways for students to be able to prove they know the material.

Crucial point, understanding the thought process of how students value their education.

Many students feel this way, and I feel this is what takes the passion out of learning. All you focus on is getting a good grade.

eLife Assessment

This study provides important findings that during credit assignment, the lateral orbitofrontal cortex (lOFC) and hippocampus (HC) encode causal choice representations, while the frontopolar cortex (FPl) mediates HC -lOFC interactions when the causality needs to be maintained over longer distractions. While this research offers compelling evidence and employs sophisticated multivariate pattern analysis, there are some concerns regarding a) task design which may have oversimplified real-world credit assignment complexities, and b) the interpretation of results. This work will be of interest to cognitive and computational neuroscientists who work on value-based decision-making and fronto-hippocampal circuits.

Reviewer #1 (Public review):

Summary:

The authors conducted a study on one of the fundamental research topics in neuroscience: neural mechanisms of credit assignment. Building on the original studies of Walton and his colleagues and subsequent studies on the same topic, the authors extended the research into the delayed credit assignment problem with clever task design, which compared the non-delayed (direct) and delayed (indirect) credit assignment processes. Their primary goal was to elucidate the neural basis of these processes in humans, advancing our understanding beyond previous studies.

Strengths:

(1) Innovative task design distinguishing between direct and indirect credit assignment.

(2) Use of sophisticated multivariate pattern analysis to identify neural correlates of pending representations.

(3) Well-executed study with clear presentation of results.

(4) Extension of previous research to human subjects, providing valuable comparative insights.

Considerations for Future Research:

(1) The task design, while clear and effective, might be further developed to capture more real-world complexity in credit assignment.

(2) There's potential for deeper exploration of the role of task structure understanding in credit assignment processes.

(3) The interpretation of lateral orbitofrontal cortex (lOFC) involvement could be expanded to consider its role in both credit assignment and task structure representation.

Achievement of Aims and Support of Conclusions:

The authors successfully achieved their aim of investigating direct and indirect credit assignment processes in humans. Their results provide valuable insights into the neural representations involved in these processes. The study's conclusions are generally well-supported by the data, particularly in identifying neural correlates of pending representations crucial for delayed credit assignment.

Impact on the Field and Utility of Methods:

This study makes a significant contribution to the field of credit assignment research by bridging animal and human studies. The methods, particularly the multivariate pattern analysis approach, provide a robust template for future investigations in this area. The data generated offers valuable insights for researchers comparing human and animal models of credit assignment, as well as those studying the neural basis of decision-making and learning.

The study's focus on the lOFC and its role in credit assignment adds to our understanding of this brain region's function.

Additional Context and Future Directions:

(1) Temporal ambiguity in credit assignment: While the current design provides clear task conditions, future studies could explore more ambiguous scenarios to further reflect real-world complexity.

(2) Role of task structure understanding: The difference in task comprehension between human subjects in this study and animal subjects in previous studies offers an interesting point of comparison.

(3) The authors used a sophisticated method of multivariate pattern analysis to find the neural correlate of the pending representation of the previous choice, which will be used for the credit assignment process in the later trials. The authors tend to use expressions that these representations are maintained throughout this intervening period. However, the analysis period is specifically at the feedback period, which is irrelevant to the credit assignment of the immediately preceding choice. This task period can interfere with the ongoing credit assignment process. Thus, rather than the passive process of maintaining the information of the previous choice, the activity of this specific period can mean the active process of protecting the information from interfering and irrelevant information. It would be great if the authors could comment on this important interpretational issue.

(4) Broader neural involvement: While the focus on specific regions of interest (ROIs) provided clear results, future studies could benefit from a whole-brain analysis approach to provide a more comprehensive understanding of the neural networks involved in credit assignment.

Reviewer #2 (Public review):

Summary:

The present manuscript addresses a longstanding challenge in neuroscience: how the brain assigns credit for delayed outcomes, especially in real-world learning scenarios where decisions and outcomes are separated by time. The authors focus on the lateral orbitofrontal cortex and hippocampus, key regions involved in contingent learning. By integrating fMRI data and behavioral tasks, the authors examined how neural circuits maintain a causal link between past decisions and delayed outcomes. Their findings offer insights into mechanisms that could have critical implications for understanding human decision-making.

Strengths:

(1) The experimental designs were extremely well thought-out. The authors successfully coupled behavioral data and neural measures (through fMRI) to explore the neural mechanisms of contingent learning. This integration adds robustness to the findings and strengthens their relevance.

(2) The emphasis on the interaction between the lateral orbitofrontal cortex (lOFC) and hippocampus (HC) in this study is very well-targeted. The reported findings regarding their dynamic interactions provide valuable insights into contingent learning in humans.

(3) The use of an advanced modeling framework and analytical techniques allowed the authors to uncover new mechanistic insights regarding a complex case of the decision-making process. The methods developed will also benefit analyses of future neuroimaging data on a range of decision-making tasks as well.

Weaknesses:

Given the limited temporal resolution of fMRI and that the measured signal is an indirect measure of neural activity, it is unclear the extent to which the reported causality reflects the true relationship/interactions between neurons in different regions.

Reviewer #3 (Public review):

The authors apply multivoxel decoding analyses from fMRI during reward feedback about the cues previously chosen that led to that feedback. They compare two versions of the task - one in which the feedback is provided about the current trial, and one in which the feedback is provided about the previous trial. Reward probability changes slowly over time, so subjects need to identify which cues are leading to reward at a given time. They find that evidence for recall of the cue in the lateral orbitofrontal cortex (lOFC) and hippocampus (HC). They also find that in the second condition, where feedback is for the one-back trial, this representation is mediated by the lateral frontal pole (FPl).

Overall, the analyses are clean and elegant and seem to be complete. I have only a few comments.

(1) They do find (not surprisingly) that the one-back task is harder. It would be good to ensure that the reason that they had more trouble detecting direct HC & lOFC effects on the harder task was not because the task is harder and thus that there are more learning failures on the harder one-back task. (I suspect their explanation that it is mediated by FPl is likely to be correct. But it would be nice to do some subsampling of the zero-back task [matched to the success rate of the one-back task] to ensure that they still see the direct HC and lOFC there).

(2) The evidence that they present in the main text (Figure 3) that the HC and lOFC are mediated by FPl is a correlation. I found the evidence presented in Supplemental Figure 7 to be much more convincing. As I understand it, what they are showing in SF7 is that when FPl decodes the cue, then (and only then) HC and lOFC decode the cue. If my understanding is correct, then this is a much cleaner explanation for what is going on than the secondary correlation analysis. If my understanding here is incorrect, then they should provide a better explanation of what is going on so as to not confuse the reader.

(3) I like the idea of "credit spreading" across trials (Figure 1E). I think that credit spreading in each direction (into the past [lower left] and into the future [upper right]) is not equivalent. This can be seen in Figure 1D, where the two tasks show credit spreading differently. I think a lot more could be studied here. Does credit spreading in each of these directions decode in interesting ways in different places in the brain?

The impact of action in the financial markets do not result in immediate outcomes. Instead actions will have underlying 'invisible' impacts that will go unnoticed for an extended time or until there is a major failure.

I don't know why I laughed at this

I get that; appreciating the silence of life

Supreme Cause?

what is it?

what is "this" exactly?

if you stop putting in effort / get lazy your ability to influence change goes away

focus on the now; don't dwell on the past or future, stay present

this also has me thinking a lot. I can't tell if its actually an impressive statement or if its just worded fancily

wow this really got me thinking

all is one

I dont know if "Self" has been capitalized this whole time but this is the first time it's stuck out to me

im wondering the same

you exist in the world, the world doesn't necessarily exist for you. I don't know this is a weird statement

why not now? why just the future?

I don't know why but I thought of the quote "to be loved is to be understood"

consistency can be terrifying but also comforting. depends on the person

why is this quoted? where is it from?

sometimes people give in

is this basically saying that when you send out your love into the distance it harms those closest to you?

sometimes the truth is hard to hear

hm I can't tell if I like this statement or not

words having an impact; hold so much truth / importance that they cannot be ignored

chased not the chaser

don't underestimate the young

who? what?

follow what has been put in place; destiny

hm what power?

be spontaneous, be yourself, be true and ignore the voices saying otherwise

dismissing own thoughts because we tend to carry the mindset that what we think is "wrong" in a way or not as important as other peoples' thoughts

connecting to wild-life and animals way of survival

I think this could mean that whatever we do, good or bad, it will follow us (like shadows) for the rest of our lives

Beat the standard truth, and prevail over it.

Omg we made it, we are peak american lit

Just do it - John Cena

Be so fr. No he did not and you saying everyone has the same blood, means you should have known better.

Its giving, "We cry, your cry, we all crew"

Just a big circle

You gott create work, not study his

Who are the aliens?

The name, wonder why he puts it almost at the end?

Kinda like Farmer James

Of the mind?

As long as your intentions are pure, do whatever you want.

Okay he kinda ate here.

Everyone is the same.

Here we go again

Move on or stay depressed.

Youth better than older generation?

God doesn't let you do/think certaint hings though so how is that self freedom?

own guide; self-reliant; in control of your own life and "destiny"

Be true to yourself, but still believe in god.

1 ruler has an immense amount of power over the lives of millions.

Seems like he really wants/likes attention. Also still no mention of women.

What if its bad? Would that change anything, or should you still be true to yourself at the expense of others?

Burn it

The law does this a lot, you can't trust only your memory.

Depends on the person

Being nice gets you way more things than being mean, just saying.

As long as you stay true to yourself you can/will be great

Again he doesn't care about others, only what is true to himself.

Would he say the same towards modern day things like charities and small buisnesses?

Because people dont like to hear the truth sometimes

Good and bad are concepts that change based on the person, I really like this.

Okay he really is proving that he will follow his own thoughts even if its the devil.

Capitalism, but it depends on the situation, nowadays people want to be different while still fitiing in.

In order to bring up issues that need to be changed you need to be able to speak them out

Magic river, reminds me of the magic mountain.

This is all in his mind.

What about the woman youth?

Young people will always have the power to change the future no matter what period you are in.

The natives?

God? If not nobody is absolutely trustworthy

Are the cowards the ones who dont believe in themselves?

If he is given something he forgets about all the other things up for the taking?

He really wants us to be ourselves no matter what.

Be who you are, and that is strengthened by the bible.

so this is nottttt what we're gonna say!

Who are they referring to as aliens...

hmmmm

If it cannot be changed, then leave it in the past and continue on, it'll change itself

Farmer James

As soon as someone is devoted to God, God will be present in their life.

Lives in the present, as Emerson said before is the way of living fully.

The perfect man doesn't need anyone other than himself.

Look within yourself, make sense considering the title

good one Emerson

lol same

this is reasonable, and I actually agree with this about many things

what is the divine fact?

which is....

its common to dwell on past experiences, but you will feel the must human. in the present.

how can God do that

Before the introduction of Lycurgus, there was extreme wealth inequality between the plebeians and the ruling elites, resulting in social tension and discord between the people of Sparta. In order for the nation to become self-sufficient and harmonious, Lycurgus implemented a drastic solution to redistribute wealth throughout through the land by dividing the land into equal portuons between the elites and the perioeci. It is an interesting afterthought after all, as it closely resembles one of the policies of an utopian society by eliminating the economic foundations of class distinction based on wealth. There was also mentions of switching gold and silver currency with that of iron, to eliminate materialism and encourage the prople to focus more on equality and austerity. Although it is an ideal theory, the real life applications of such theories has too many limitations that stops it from becoming an ideal method to achieve equality. Past explorations into enforcing a state of economic equality throughout modern history has failed, such as the implementation of collectivism throughout the USSR and Post WW2 China. The juxtaposition between soviet collectivisation and Spartan Syssitia is clear, one focuses on trying to provide and distribute food to the masses, while the other one's main focus is to enforce equality. The irony, unfortunately, is clear. Both of them, in the effort to eliminate hierarchies, inadvertently created new ones.

The text emphasizes that learning should extend beyond the classroom to include adults and activists from the local community. This points to the importance of connecting students with real-world examples and mentors who can enrich their educational experience. The statement about students feeling valued for their ideas, attitudes, and skills highlights the psychological and emotional aspects of education.

The text opens with a shift in the educational environment, signaling a change in the author's experience with Ms. Hill's class, suggesting that the dynamics of classroom discussions can significantly impact students' emotional and academic well-being

The text starts with the assertion that the school system privileges individuals who align with the dominant culture, which is typically associated with middle-class and upper-middle-class values and behaviors. This points to systemic biases within educational institutions that favor certain cultural norms.

The text identifies that there are fewer stereotypes regarding academic success for Latino and African American students. This observation highlights a disparity in societal perceptions of different racial groups. The text states that the absence of stereotypes for Latino and African American students can lead to a lack of recognition of their hard work and achievements.

The text points out that there are fewer stereotypes surrounding academic success for Latino and African American students compared to their White and Asian American counterparts. This absence can lead to a lack of recognition of the hard work and achievements of these communities.

Title should be verify state of an object

This syntax is wrong there is no control here it should be: Verify the value of "John" Text is within ...

This example is wrong as it shows an icon with an associated text. it should be: Verify the alarm exists This also brings the question of the syntax since the <text for identification> should be optional

from : Custom Apps Drive HTTP actions peergos book

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Stomach ulcer Prevacid is indicated for the treatment of patients with H. pylori infection and duodenal ulcer disease (active or one year history of a duodenal ulcer) to eradicate H. pylori

HE SMOKES! what does "he thinks he eats good" means...

Unstable angina (probably no longer stable) Myocardial Infarction Pulmonary Embolism, Coronary Embolism, etc (more unlikely because they tend to cause shortness of breath which the patient doesn't have) GERD

This text highlights the critical issue of educational inequality and the ways in which institutional structures can perpetuate disparities among students. The advantage enjoyed by students from private feeder schools raises questions about fairness and access to high quality education.

The text further illustrates the flexible tracking system by detailing the options available to ninth graders for foreign language electives. This text highlights the evolving nature of tracking in American high schools, emphasizing the balance between maintaining academic standards and providing students with greater flexibility and choice.

The text acknowledges that schools have limited ability to change the inequalities in students' backgrounds, such as socioeconomic status or family circumstances.

The text highlights a contradiction between the school’s focus on diversity and the ongoing achievement gap. Berkeley High is described as having institutional features, such as courses and departments that celebrate diversity, yet these efforts are not translating into equal academic success for all students.

Due to the apparent issues with this type of thinking, some changes in common pedagogy have occurred since then. As someone with no prior computer-science knowledge, I have seen that this course is far more focused on thinking about the art of problem solving, then it is about the logic of machines. We are able to atomically break down the way our programs work to the computer with the stepper, but just as often it's useful instead to undergo a design process. We think about what we want to achieve, and break it down into goals a human finds intuitive, rather than focusing on the thought process of the computer.

Parnas says that it is unlikely that research towards automatic programming is going to bring real results. However, with the creation of public AI such as ChatGPT for public use, you could say that the military has better AI than the public. Thus, the military's automatic programming has significantly improved from his time.

Parnas says that they do not expect A.I to build military software, and while they may not use it entirely, they likely will use it to some degree. Artificial intelligence is getting smarter everyday and keeps showing more and more power with each and every update. One day we ask "I wonder if it will be able to do this" and the next update it just that with ease. A.I is getting much stronger than ever before and will likely be used in military software if it is not already.

As software development/programming has grown as a discipline more standardized learning methods have become available, leading to better education outcomes and expected knowledge base.

Since 1985, AI has vastly improved in terms of reliability and understanding of program behavior. Modern AI systems use complex machine learning models rather than simple rule-based heuristics. Techniques like neural networks and reinforcement learning have made it possible for AI to handle tasks like image recognition, speech processing, and even medical diagnosis with much greater accuracy and predictability than the heuristic-based AI of the 1980s.

Getting inspired by things he encountered at the cemetery is an excellent idea. I liked that and I am planing to use it in the future.

Great start of describing writing place environment and how change based on the writer state of mind

Gawain's reputation holds a lot of weight both with Lady Bertilak and Lord Bertilak, but in different contexts. The Lady's image of Gawain lies in courtly manner. The Lord's image of Gawain lies in his brave, honest, and other knightly attributes. Both of which he must uphold. The modern equivalent is having our reputation precede us and feeling pressure to uphold our image or face the consequences of failing.

Aside from christian discourse, the hyper fixation on male homosexual relation in comparison to female homosexual relations is extremely present in the modern world. Perhaps it has something to do with societal norms and how cultures view masculinity. To be masculine is to refrain from emotional and physical expression of love, care, and tenderness. Tenderness and softness are expected traits of femininity, maybe this is why it's so culturally consuming to see two men engage physical and emotional romance.

The interpretation of a kiss between men can greatly vary depending on the cultural context. I argue that if a 21st century, american, heterosexual, male were to be transported into the medieval period and integrated into society, he would be able to become accustom to kisses between men as greetings, homage, or parting of ways. It is easier to adapt the the dominant cultural norms rather than vice versa.

The polar spirit is not an actual character, more of a presence. A looming presence throughout the reading that punishes the Mariner and makes sure to carry out his curse of suffering. LS

The Albatross is a creature of love and grace that was introduced to the sea men. It symbolizes innocence and love, and it is believed to be symbolism for Jesus Christ. It brings many happy gifts to the sailors, but it is killed by the mariner. LS

The Mariner's crew are an impressionable bunch, they are originally captivated by the Albatross, but only for what it could provide and not the creature itself. They are indifferent to its killing and only regret it because they can no longer reap the rewards of it's presence. LS

The Wedding Guest is a listener to the Mariner's story, they are perhaps seen as naive and in need of this story to gain the experience to witness a wedding. They leave the story feeling wiser. LS

The story's protagonist. An old sailor who is perhaps jaded and didn't have much hope left even prior to meeting the Albatross. After inevitably killing the Albatross he is cursed to forever be tortured and forced to tell his tale. LS

Theme (and message) of treating all lives with love and kindness, which comes from the other theme that all lives are valuable, since they were created by God. -MH

Theme of redemption and forgiveness, specifically the redeeming and healing power of prayer/God. -MH

Theme of being haunted by guilt/past sins, as well as the Isolation, Fear, and Control that the continued memory of its causal event still brings/has upon its sufferer's life, even after progress towards redemption has been made. This also connects with the theme of redemption itself, as it is that shadowing regret that compels one to overcome and dissipate it through redemption.

Also, as a fun, semi-related note, this stanza was repurposed in Frankenstein. -MH

One example I can think of is TikTok and Xiaohongshu. Both have graphic sharing functions. But the next steps are slightly different. While TikTok focuses on fostering group chats and building connections between different users, Xiaohongshu follows up by guiding users to post comments and chat individually.

The narrator's feelings change from giving money to feeling hatred. This shows how past relationships can be complicated and filled with regret.

The music is a rare moment of beauty in a harsh world. It shows that even in tough times, there’s a desire to express feelings and find joy.

The laughter is mean and not joyful, showing how tough and unkind their environment is. It highlights the sadness and challenges children face.

This is one of my favorite quotes from the story. This is because it is true that anyone can get "sucked under", not just specific types of people.

The heading and first three lines of this section are an intricate reworking of the scene of the death of Jesus Christ in the Bible. In order to understand this collage of biblical material, the reader must first allow the title of the section to become the first line of the poem, which is grammatically sound as an antecedent before the list of prepositions in the following three lines beginning with “after.” With this in mind, we can turn to “thunder” as it appears in the heading for this section, where Eliot seems to render it its own character in the poem. We can identify the “thunder” as some sort of divine being. After Jesus’s death in the Book of John, it is written that “The crowd that stood there and heard it said that it had thundered” (John 12:29). Presumably, the thunder occurred by the hand of God, or represents the voice of God. In fact, Eliot’s God might represent more than just the Christian God. In Themis’s essay, she claims that “the thunderbolt was to the primitive Greek not the symbol or attribute of the god, but itself the divine thing, the embodiment and vehicle of the god” (Themis, 62). Given both religions justify the thunderbolt as a celestial voice of sorts, we continue to the following lines, which appear to take the form of the chronology of the death of Jesus as it appears in the book of John. First, the “torchlight” in “The Waste Land” surely references the Pharisees who Jesus himself describes as holding “torches” (John 18:3). In the second line, the “frosty silence in the gardens” is reflected in the biblical text when Jesus takes his “disciples over the brook Cedron, where was a garden” (John 18:1), the garden which is described as “cold” (John 18:18). “Silence” may refer to Peter when he “smote the high priest's servant, and cut off his right ear” (18:10). Then, “stone agony” likely nods to the flogging of Jesus in John 19:1. Given the grammatical mechanics of these lines, it appears that the setting of “What the Thunder Said” is Jerusalem, when the thunder sounded right after Jesus died. In other words, perhaps the text that follows is through the voice of God, an interesting lens through which to approach the section.

In his annotation from last year, Parth draws a very interesting parallel between the onomatopoeic dripping of water and atonal music: “this onomatopoeia…lacks a concrete framework with which the notes—"drip" and "drop"—arrange themselves, nor does it have a "triad" that the notes "drip" and "drop" must return to.” Parth goes on to interpret the atonality of water as related to purity and constancy. Building on-bending, if you will-Parth’s analysis, an interesting added dimension here is that, if the grounded reality of this section is atonal, the portions where water is hypothetically present induces a more perplexing state because we are goaded with the almost tangible yet unrealizable presence of abundant water. Music traditionally has key because it is pleasant and reassuring for eon-conditioned human ears and consciousnesses. So, by creating tension between atonally dripping water and the promise of abundant water, Eliot casts the presence of water as a more abstract and less certain entity.

This interpretation has some logical basis because, unlike atonal music which is characterizable despite being disorienting at first, the water here only exists in the mind of the speaker who is yearning for hydration, ostensibly beyond the absolute comprehension of even the most astute scholar in a similar way to how the water is beyond the speaker’s scope of consumption. This taunting absence is extended even to sweat, which is somehow “dry” and “sterile” thunder which offers the promise of torrential rain and salvation but never actually offers such a luxury. Also, in analyzing the promise of water, the contrast between it–the most essential substance for human life and rocks–rather useless objects–is also quite interesting. Rocks are quite abundant but “Amongst the rock one cannot stop or think.” Thus, at an obvious level, a broader interpretation here is that Eliot is highlighting the criticality of certain substances to human existence and vividly illustrating the unique torture of not having access to them through the creation of a hypothetical. However, an added layer of nuance can be unlocked by contrasting Eliot with Keats.

Of course, Keats’ work is a more optimistic one in that it casts nature as offering realizable solace while Eliot creates a dynamic verging on torture. More interesting, however, is the contrasting messages on renewal that emerge. Despite not necessarily providing detailed reasoning, indeed rejecting convoluted thought processes generally–as he writes to “fret not after knowledge–I have none”–Keats suggests the presence of some larger force that will assure renewal and ensure that “spring will be a harvest-time.” Eliot, on the other hand, offers an abundance of twisting, looping, and otherwise confusing thoughts on water’s presence, or the lack thereof, and by extension suggests that renewal, which requires water, is not guaranteed. In TWL, there is only the rawness of nature and the speaker, no larger force that allows one to exist in the bliss of having no knowledge and believing in external forces.

Tying this message on optimism back into my preceding analysis on Eliot’s gauding with water in a wasteland where there is no water–interspersing comfort-inducing tone-into atonal music–the more profound commentary of this section seems to be that humans exist in an inherently frail state where the lack of even the simplest substance of water–that is taken for granted with increasing frequency as development grows–can render us impotent and open to the emotional manipulation that the speaker in this section suffers as he yearns for water, a reflection of his physical suffering.

The belief that humans are meant for greater pursuits intertwines with the inevitability of death. Despite Eliot’s lack of appreciation for Tennyson’s narrative, this idea seems to have been inspired by Tennyson’s poem. The final line of “Ulysses” becomes the most striking and seemingly summarizes its entire purpose as Ulysses states “to strive, to seek, to find, and not to yield.” For Ulysses, life’s meaning lies in this quest for knowledge and purpose, despite the certainty of death.

Dante’s description offers a deeper insight into this theme. When Ulysses describes his story he explains how he encouraged his comrades to embark on a journey, declaring “you were not made to live like brutes or beasts, but to pursue virtue and knowledge.” The comparison to inhumane and violent creatures further signifies a human aspiration to seek meaning beyond mere survival. This pursuit, however, is deemed ineffective: as Ulysses describes, “the whirlwind" stroke the boat “the sea closed over” them, describing how human ambition becomes futile in the face of inescapable fate.

Eliot reiterates the same theme for Phlebas, who is merely a product of Eliot’s imagination. The readers have no idea of the life of the character and it never becomes important: this meaningless life of a made-up character, however, leads to the same outcome, which Eliot underscores by invoking a similar image of a whirlpool. Furthermore, Eliot’s choice to introduce a character without a rich backstory underscores this notion: all lives, regardless of their perceived significance, converge on the same fate, where death, as an unyielding current, eventually claims every life. A change from whirlwind, which is caused by instability of wind can happen anywhere, to whirlpool, which are results of an intersection of two opposing currents, becomes particularly interesting. Instead of focusing on unpredictability, Eliot uses this image to highlight the result of opposing a sort of “life’s current.”

The title of this section, a reiteration of Madame Sosostris’s prophecy to “fear death by water,” adds a layer of irony to this opposition: what is the point of fearing this death if Eliot had already predetermined it, had already written both the readers’ and the characters’ fates within the poem? This pathology serves as a direct analogy to reality, where no matter the fear or attempts to battle the circumstances, the outcome remains unchanged.

In a sense, it appears that Eliot encourages readers to resign to circumstances, as he contrasts the previous Fire Sermon with this Death by Water. The struggles, dissociations, and pleas of the Fire Sermon are juxtaposed with the calm detached description of the Death by Water.

In contrasting these two sections, Eliot presents two different responses to existential challenges. Whether with Philomela, who attempts to voice her pain as a nightingale with “tereu,” however, as a female nightingale is unable to produce sound, the unnamed female who desperately asks her partner “Why do you never speak to me,” or the five burnings that might relate to disconnection from the five senses in response to the character’s final plea: “O lorg thou pluckst me out”, the characters in "The Fire Sermon" grapple with external circumstances. Whether relationships, disconnection, or the chaos of modern life, their attempts to fight against these forces prove ineffective. Instead, their search for meanings only reveals the futility of their resistance.

In contrast, "Death by Water" offers a sense of acceptance of mortality. Phlebas has been dead for two weeks – a “fortnight,” when there is nothing to be done anymore. Instead, external circumstances, like “a current under the sea,” carry his body. Eliot reiterates its overarching nature, drawing a comparison between Phlebas, “Gentile or Jew” and the readers.

The effectiveness of this acceptance is further reflected through form: Death by Water becomes straightforward and concise, while the Fire Sermon constantly grapples with dissonance, contrasting voices, and changes in form.

Whether in life or in death, external circumstances, like a “current under the sea” seem to carry us to the final destination that remains unchanged despite the life pursuits. Death by Water, thus, invokes a question: maybe it is in the acceptance of the certainty of death and refusal to fight these external circumstances that lies a potential for peace amid this chaos of life?

"Pornography" as in books about LGBTQ+ people?

The text acknowledges the difficulty in precisely determining the effects of various family-related factors on children's school readiness. The text asserts that the rise in income inequality since the 1970s and the concurrent widening gap in school success between children from different income levels is not a coincidence.

The text also highlights the importance of family structure in shaping children's academic trajectories. Both Anthony and Harold were raised primarily by single mothers, a situation that is common in low-income families but rare in high-income families.

The text begins by discussing the impact of income inequality on children’s educational success. The circumstances that Anthony and Harold grew up in, left them at a disadvantage compared to children from wealthier families. Although some inequality has always existed, the income gap has widened significantly over the past four decades, which has exacerbated disparities in educational opportunities and outcomes.

The text emphasizes that even modest income increases can lead to measurable improvements in children’s achievement test scores, raising them by the equivalent of about 20 SAT points. The text suggests that increasing income may be one way to help narrow the achievement gap.

Income is a major factor in determining a child's academic success. National studies consistently show that children from higher-income families perform better in school, and the achievement gaps between wealthy and low-income children have grown significantly over time. These gaps highlight the powerful influence of socioeconomic status on educational outcomes

The bar chart in figure 3.1 visually represents the magnitude of income-based achievement gaps. This quantitative approach illustrates how substantial these gaps are, emphasizing that income plays a powerful role in determining educational success.

The text highlights that beyond academic skills, children from higher-income families tend to have advantages in behavioral and emotional well-being. They are more engaged in school, less likely to engage in antisocial behavior, and less likely to struggle with mental health issues compared to their lower-income peers. These behavioral and emotional differences still contribute to overall school success and remain stable throughout elementary and high school.

The use of a "100-point difference" to represent one standard deviation allows for the measurement of how much high- and low-income children's achievements differ over time. The reference to figure 3.1 suggests that the size of the gap between high- and low-income children is significant, underlining income as one of the most powerful factors influencing academic success.

The text points to how socioeconomic factors such as family income, parental education, and family structure can significantly shape a child’s future, perpetuating cycles of inequality

I did not know that. No wonder clocks have been so relevant through the centuries.

I find it interesting how there were multiple languages used amongst Rome. Making the use of languages very diverse. From lower classes to upper classes.

When colonizers forced Indigenous peoples and African peoples not to speak their languages, that imapcted the world more than we understand

It is amazing how much culture impacts our outlook on life and how we respond to events, big or small

Wouldn't that be considered a scroll, rather than a book?

Mechanics to this day are crucial to our society.

I am curious as to how water was used in order to abstract golds.

I can understand as to why clocks are heavily referenced, mainly because of their longevity of relevance.

I find it super interesting that an invention from way back then, is still essential in todays society.

DUDE...Fucking B cell depletion CAR T therapy to cure the autoimmune disease. Autologous stem cells to regenerate the organ.

HOLY SHIT

WOW..... That's a cure to AA disease.

Very good example! It swiftly depicts the correlation between Du Bois's charts and photographs, how they work together to uncover a panoramic view of individual lives of black people for us.

There is an intent of causing fear and harm to the reader and they are trying to convince them of a claim that is not backed up by science and the research behind that science.

This tone and wording indicates that the author does not fully know the information/misinformation they are writing and that causes uncertainty to the reader.

This is an example of disinformation. One could tell that this information is disinformation because it is an unreliable source that does not have proper knowledge behind their argument. This opinion and disinformation manipulates a public opinion with not much thought and research behind. This was very identifiable as disinformation because of the informal tone and word usage.

The conduct of a criminal prosecution and the decision to investigate whether to prosecute involves the exercise of executive power, but it is not purely executive in nature.

This tone and wording indicates that the author does not fully know the information/misinformation they are writing and that causes uncertainty to the reader.