Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
Ritzau-Jost et al. investigate the potential contribution of AP broadening in homeostatic upregulation of neuronal network activity with a specific focus on dissociated neuronal cultures. In cultures obtained from a few brain regions from mice or rats using different culture conditions and examined by different laboratories, AP half-width remained stable despite chronic activity block with TTX. The finding suggests that AP width is not significantly modulated by changes in sodium channel activity.
Strengths:
The collaborative nature of the study amongst the neuronal culture experts and the rigorous electrophysiological assessments provides for a compelling support of the main conclusion.
Weaknesses:
Given the negative nature of the results, a couple of remaining issues (such as the cell density of cultures and the presentation of imaging experiments with a voltage sensor) warrant further consideration. In addition, a discussion of the reasons for the I stability of AP half-width to sodium channel modulation might help extend the scope of the study beyond the presentation of a negative conclusion.
We would like to thank the reviewer for positively evaluating our manuscript. Please find below our detailed point-to-point response to the reviewer’s comments.
Reviewer #2 (Public review):
Summary:
This study reexamined the idea that action potential broadening serves as a homeostatic mechanism to compensate for changes in network activity. The key finding was that, while action potential broadening does occur in certain neurons - such as CA3 pyramidal cells-it is far from a universal response. This is important because it helps resolve longstanding discrepancies in the field, thereby contributing to a better understanding of network dynamics. The replication of these findings across multiple laboratories further strengthened the study's rigor.
Strengths:
Mechanisms of network homeostasis are essential to understand network dynamics.
Weaknesses:
No weaknesses were noted by this reviewer.
We would like to thank the reviewer for the positive evaluation of our manuscript. Please find below our detailed point-to-point response to the reviewer’s comments.
Reviewer #3 (Public review):
Summary:
The manuscript "Unreliable homeostatic action potential broadening in cultured dissociated neurons" by Ritzau-Jost et al. investigates action potential (AP) broadening as a mechanism underlying homeostatic synaptic plasticity. Given the existing variability in the literature concerning AP broadening, the authors address an important and timely research question of considerable interest to the field.
The study systematically demonstrates cell-type- and model-specific AP broadening in hippocampal neurons after chronic treatment with either tetrodotoxin (TTX) or glutamatergic transmission blockers. The findings indicate AP broadening in CA3 pyramidal neurons in organotypic cultures after TTX treatment, but notably not in dissociated hippocampal neurons under identical conditions. However, blocking glutamatergic neurotransmission caused AP broadening in dissociated hippocampal neurons. Moreover, extensive evaluations in neocortical dissociated cultures robustly challenge previous findings by revealing a lack of AP broadening following TTX treatment. Additionally, the proposed role of BK-type potassium channels in mediating AP broadening is convincingly questioned through complementary electrophysiological and voltage-imaging experiments.
Strengths:
The manuscript exhibits an outstanding experimental design, employing state-of-the-art techniques and a rigorous multi-lab validation approach that greatly enhances scientific reliability. The experimental results are meticulously illustrated, and the conclusions drawn are justified and supported by the presented data. Furthermore, the manuscript is comprehensively and clearly written.
Weaknesses:
Concerning the statistical analyses employed, it is advisable to consider the Kruskal-Wallis test with corrections for multiple comparisons when evaluating more than two experimental groups.
We would like to thank the reviewer for the positive evaluation of our manuscript. In the following we first address the comment regarding the used statistical tests. Please also find below the detailed response to the reviewer’s further comments. Indeed, we did not apply a correction for multiple comparisons in Figure 2. This seems justified because in this exceptional case we are more worried about type II errors (false negative). The Kruskal-Wallis test seems not appropriate for this type of data for which only the comparison between the control and respective TTX data is relevant. Instead, we followed the reviewer’s suggestion by applying corrections for false discovery rate (FDR). We thank the reviewer for pointing out this statistical issue and addressed it in the revised manuscript (lines 121–128):
“Even though AP durations varied up to 2-fold between conditions, statistically significant homeostatic AP broadening was not detectable in any of the tested conditions (Fig. 2B). To minimize type II errors (false negative) we intentionally did not apply a correction for multiple comparisons. The only significance was observed in condition III but in an opposite direction (i.e. AP narrowing with TTX, P=0.026; Fig. 2B). However, this is likely a false positive because application of corrections for false discovery rate results in P=0.268 for both Benjamini–Hochberg and Bonferroni correction.”
Recommendations for the authors:
Reviewing Editor Comments:
The main and most important observation of the study is that the AP does not change in most cases examined. A discussion of the mechanisms of the changes in CA3 neurons would significantly strengthen the compelling evidence presented. The individual reviews are also provided, in case the authors find them useful to include other aspects suggested by the reviewers.
We would like to thank the Reviewing Editor for handing our manuscript and for the positive evaluation of our work. The main focus of our study was the analysis of homeostatic plasticity in cultured neurons of the neocortex. We agree that the findings in CA3 neurons are interesting. As explained in more detail below, we have carefully discussed the mechanisms of the changes in CA3 neurons in the revised manuscript.
Reviewer #1 (Recommendations for the authors):
Major points
(1) AP widths measured in the present study under basal conditions are generally larger than the value reported in previous work by Li et al. 2020 (~1.5 ms). In particular, rat cortical cultures prepared using the same conditions show that the mean AP half-width in controls of the present study (~2.5 ms) is closer to the mean AP half-width in TTX-treated neurons in Li et al. (~2.0 ms).
We thank the reviewer for the detailed and positive feedback as well as for the thoughtful questions. The inconsistency of action potential half-duration reported in our and Li et al.’s data is partially due to differences in the way the half-duration was measured. In Li et al. the exact method is unfortunately not defined, but from a personal communication with the authors we know that they measured half-duration based on the AP amplitude between AP peak and AP voltage threshold. In contrast, we measured half-duration based on the AP amplitude between AP peak and the resting membrane potential preceding current injections. When we measure AP half-duration instead from voltage threshold, the average half-durations are 1.97 ms (compared to 2.64 ms from baseline, n = 106 cells; average across conditions I–IV, control and TTX merged). Thus, the discrepancy in the half-duration is to a significant proportion due to methodical differences in the way the half-duration was measured.
One parameter that is not stated in either study is cell plating density, which can potentially bias the neuronal network activity levels of cultures. Could the authors comment on the possible contribution of neuronal culture density to AP half-width under basal recording conditions and its sensitivity to chronic TTX treatment? Are there any data available? For example, cultures used by Li et al may have been plated at a high density and experienced high activity level during culturing, which could have contributed to the enhanced sensitivity to chronic activity suppression by TTX.
We agree that neuronal culture density is an important factor influencing neuronal activity and hence potentially also the sensitivity to chronic activity suppression. In our experiments, the number of plated cells per cover slip varied between conditions about 3-fold: 30–50k cells for conditions I and II, 25–30k cells for conditions III, VII, XI, 50k cells for condition IV, 65k for conditions V, VI and VIII, and 70k cells for conditions IX and X. Li et al. do not provide the cell density or the number of plated cells. Despite the difference in the number of plated cells in our dataset across various laboratories, we did not observe a systematic effect of cell number on baseline AP half-duration. Furthermore, we observed strongly different baseline activity across our various experimental conditions (Fig. 3A), which did not correlate with cell density. Also, we did not notice an impact of baseline activity on the sensitivity to chronic activity suppression with TTX (cf. Fig. 3A and 2B). We have now added the number of plated cells per condition to the methods section as well as the following paragraph to the discussion section (lines 256–262):
“The sensitivity to chronic TTX treatment might depend on baseline neuronal activity, which is in part related to neuronal culture density[37]. However, TTX did not induce AP broadening despite different baseline activities (Fig. 3A) and a nearly threefold variation in the number of plated cells per cover slip between conditions (25k – 70k cells per coverslip).”
In addition, a discussion of the reasons for the seeming stability of AP half-width to sodium channel modulation might help extend the scope of the study beyond the presentation of a negative conclusion.
We thank the reviewer for this suggestion and have added a paragraph to the end of the discussion emphasizing potential advantages of cell-type specific AP broadening (lines 353–362):
“Despite the lack of homeostatic, TTX-induced AP broadening in dissociated cultures, AP duration was broadened upon Kyn-treatment in dissociated cultures and using TTX in CA3 neurons in organotypic cultures. Because BK-channels control AP duration in CA3 neurons of organotypic cultures[79], homeostatic BK-channel downregulation as proposed by Li et al. may be involved in AP broadening in this specific cell type. While the reasons for the variable occurrence of homeostatic AP broadening remain unknown, this may render neuronal circuitries more robust to perturbations. The regulation of AP duration therefore might represent one element in the repertoire of neuronal plasticity that is, similar to other plasticity mechanisms, not generally shared, but specifically expressed in some cell types and neuronal compartments.”
(2) In this study, CA3 neurons in organotypic cultures were the only cells that showed AP broadening with TTX treatment. Notably, CA3 neurons show strong recurrent activity in general and would be expected to have experienced high levels of activity in culture. For CA3 neurons in organotypic cultures, does IbTx increase basal AP half-width?
We thank the reviewer for this interesting idea. Even though, to our knowledge, there is no study investigating the effect of IbTx on AP width in CA3 neurons of organotypic cultures, Raffaelli et al. (DOI 10.1113/jphysiol.2004.062661) reported ~15% AP broadening using the BK-channel blocker paxilline. Therefore, TTX-induced broadening in CA3 neurons might be related to BK-channel-dependent AP repolarisation, consistent with the model proposed by Li et al. Because organotypic cultures show increased activity for longer cultivation periods and higher connectivity compared to acute slices (De Simoni et al., DOI 10.1113/jphysiol.2003.039099), the effect of TTX may be aggravated in organotypic cultures compared to acute slices or in vivo. However, the lack of a TTX-effect was not dependent on background neuronal activity or culture density in our recordings (see above as well as lines 306–310 of the revised manuscript).
(3) Figures 4E-G. In experiments to test the efficacy of IbTx with GEVI, larger fields of view of neuron(s) used for recordings should be included. As shown, it is difficult to discern the quality of the preparation and does not provide a representative indication of the type of signals measured.
We thank the reviewer for this suggestion and have included an image of a representative neuron expressing the GEVI in Fig. 4E.
Minor points
(1) Lines 222-228. With respect to cell-type specificity of TTX-induced AP broadening, the observed lack of effect of TTX in dissociated hippocampal cultures might suggest that the cultures are predominantly DG granule cells and CA1 neurons, with few CA3 neurons surviving. Could the authors comment?
We thank the review for this interesting hypothesis and have discussed it in the manuscript as a potential explanation for our different findings in the hippocampus.(lines 263–270):
“Although we mainly focus on neocortical cultured neurons (condition I to VIII, Fig. 2) because Li et al. used neocortical neurons, the absence of AP broadening in hippocampal neurons (group IX to XI) could in principle be explained by the selective loss of CA3 neurons, which show AP broadening in organotypic cultured neurons (Fig. 1A and B). However, CA3 neurons were shown to survive in dissociated cultures following region-specific microdissection[40], and CA1 neurons are generally more stress-sensitive to excitotoxicity with glutamate or NMDA than CA3 and DG neurons[42], arguing against a general selective loss of CA3 neuron in dissociated cultures.”
(2) Figures 3D, E. To what extent is the observed increase in sEPSC amplitude due to an increase in sEPSC frequency? Is quantal amplitude increased following TTX treatment, a postsynaptic strength parameter that one would not expect to be affected by a change in AP width, but that is known to undergo up-scaling with chronic TTX treatment?
We would like to thank the reviewer for the question. We cannot rule out an interplay between sEPSC amplitude and frequency. We did not measure quantal amplitude in the presence of TTX. Our experiments were designed to test whether TTX successfully induced homeostatic plasticity, but not to attribute the observed effect to pre- and postsynaptic mechanisms. We have added the following statement to the revised manuscript, to highlight the possible interaction of sEPSC amplitude and frequency (lines 176–178):
“These changes in sEPSC amplitude and frequency are not specific for somatic, pre- or postsynaptic adaptations. However, the results show that blocking AP firing with TTX successfully induced homeostatic plasticity under our experimental conditions.”
(3) Line 132. Could the authors explain the rationale for using AP amplitude as a measure of neuronal "viability"?
In a response to Cell, Li et al. suggested that the lack of a TTX effect was due to recordings from unhealthy neurons and that small AP amplitudes could indicate impaired cell viability. Indeed, we also believe that cells which appear morphologically less healthy tend to have small and slow APs. A mechanistic rationale could be a change resting membrane potential or changes in the expression of voltage-gated sodium and potassium channels. However, AP amplitudes were not affected following TTX treatment in any of the eleven recording conditions (Fig. 2D) or a cross-conditional comparison (Fig. 2E). In the revised manuscript, we have now added a possible rationale (lines 134–137):
“Because unhealthy neurons tend to have small and slow APs, possibly due to changes in resting membrane potential or expression of voltage-gated sodium and potassium channels, we first analyzed AP amplitude as a measure of neuronal viability.”
Reviewer #3 (Recommendations for the authors):
I propose addressing the following questions, either through additional experiments (recommended) or a deeper theoretical discussion:
(1) Since the authors demonstrate that blocking glutamatergic neurotransmission in dissociated hippocampal neurons causes AP broadening, do similar phenomena occur in organotypic cultures and dissociated neocortical neurons?
We thank the reviewer for the interesting question. In dissociated hippocampal cultures, we show that AP duration is maintained following treatment with TTX and NBXQ, while Kyn-treatment leads to AP broadening (Figure 1C). To our knowledge, the effect of Kyn on AP duration has not been studied in neocortical dissociated cultured neurons. However, Kyn induced AP broadening in CA3 neurons of hippocampal organotypic cultures (Zbili et al., DOI 10.1073/pnas.2110601118) while CNQX did not induce such broadening in CA1 neurons (Karmarkar and Buonomano, DOI 10.1111/j.1460-9568.2006.04692.x). Both findings are in accord with our recordings from dissociated hippocampal cultures. These data however do not allow inference as to whether AP broadening is a cell-type specific or blocker-specific mechanism in hippocampal organotypic cultures. Because the main focus of our study is the absence of AP broadening in neocortical cultured neurons as described by Li et al., we adjusted the corresponding discussion section (lines 299–322)
“In contrast, APs were not significantly broader following synaptic block by NBQX (Fig. 1C, D), in accord with recordings from CA1 neurons in organotypic cultures using CNQX. TTX-induced broadening may therefore be cell-type specific or due to a differential effect of the glutamate receptor blockers on NMDA receptors which are blocked by Kyn but not NBQX/CNQX or TTX and which have recently been demonstrated to be important for the induction of synaptic homeostatic plasticity[41].”
(2) Are BK channels involved in AP broadening observed in CA3 pyramidal neurons in organotypic cultures?
We thank the reviewer for the question. BK channels control spike duration in CA3 neurons of organotypic cultures (~15% broadening upon block by paxilline; Raffaelli et al., DOI 10.1113/jphysiol.2004.062661). Even though there is no available data on the contribution of BK channels to homeostatic spike broadening in this cell type, CA3 neurons in organotypic cultures thereby fulfil the two necessary preconditions of the model proposed by Li et al. (namely, the control of the resting AP width by BK-channels and TTX-induced AP broadening). We include this possibility in the discussion (lines 355–357):
“Because BK-channels control AP duration in CA3 neurons of organotypic cultures[79], homeostatic BK-channel downregulation as proposed by Li et al. may be involved in AP broadening in this specific cell type.”
(3) AP broadening consistently occurs in CA3 neurons within organotypic cultures; what molecular or cellular mechanisms underpin this phenomenon, and is there a potential contribution from glial cells?
We thank the reviewer for this interesting question. CA3 neurons show AP broadening upon chronic inactivity across various studies that has not been observed in CA1 or DG neurons. Recordings from CA3 neurons served as a positive example for TTX-induced AP broadening in our study, in contrast to a lack of broadening in dissociated (neocortical and hippocampal) cultured neurons. The discrepancy between the results in dissociated and organotypic cultured neurons could indeed be due to interactions with glia cells. We have added this possibility to the discussion in the revised version of the manuscript (lines 270–273)
“Altered cell-cell interactions with glia and neurons in organotypic and dissociated neuronal cultures could instead contribute to the different findings in various hippocampal preparations.”
