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  1. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    The paper asks the question “how many types of calcium channels exist in neurons?” They make a claim that this question was fundamental in understanding how calcium entry contributed to diverse neuronal functions. They were also trying to understand the single-channel properties as well as their responsiveness to dihydropyridine compounds used in calcium channel purification. Here they report evidence for the coexistence of three types of calcium channels in sensory neurons of the dorsal root ganglion of a chick. They identified large conductance channel (L-type Ca channels) contributing long-lasting current at strong depolarizations, T-type Ca channels, a relatively tinier conductance that underlies a transient current activated at weak depolarizations, and N-type calcium channels which were neither T nor L. Lastly, they reported that the dihydropyridine calcium agonist strongly increased the opening probability of L, but not T or N-type channels. They relate their findings to be useful for the elucidation of the physiological roles of various channel types through determination of the cellular distribution and their sensitivity to neuromodulators.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    Throughout the study, they utilized electrophysiological approaches like whole cell patch clamping to kinetically distinguish components of calcium channel by applying depolarizing test pulses at various levels from different holding potentials (h.p.). This provided evidence for the basis of the existence of the three distinct components of calcium channel currents under ionic conditions that minimize contamination by other currents. They also recorded from cell-attached patches to evaluate single-channel current; recorded before and after the addition of Bay K 8644, a dihydropyridine calcium agonist. The methodological approaches used in this study were pertinent to the kind of research and proposed question to be answered. However, they could have performed a better job in the way they chose to organize and communicate the information/results.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    I think the paper doesn’t do a sufficient job at explaining the rational or scientific premise of the study. It doesn’t give a sufficient amount of information/background to be able to understand the paper, or even some of the techniques being used in the study. The paper reads like a continuation of previous work performed in the lab… so it haphazardly jumps into the questions being asked without helping their audience understand how it is of importance and why understanding its importance can help future studies; which would be a requirement were it to be published today. The way it is written overall does not encourage communication or understanding from a layman’s point of view which I think should always be the goal of scientific articles. To convey information in an understandable manner regardless of how complex the techniques being used are. In my opinion, I think it of grave importance that the authors needed to make the information less complex particularly to people who will need to fund further scientific studies such as this. The way the information relayed in the article is, it may be more suited for seasoned electrophysiologists who have spent years studying it.
    In addition, the organization of the article was also lacking in terms of the methods section or the introduction; however, the progression of the results and discussion were thorough and strongly supported. Their results and subsequent conclusions were consistent and were related with some findings in literature, while also making novel discovery on the coexistence of three types of calcium channels in one preparation. Lastly, I think the paper is lacking in discussion of what their intended future directions are, as well as the larger impact of their conclusions. They need to expand more on the “why is this important?” and not just “hey here is what we found!”.

  2. Student Aminatta Tejan-Kamara Instructor Dr. Akbarali Date 10/10/19
    Please answer the following. Do not exceed 1 page for your answer; you may use outline/bullet points.
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    Hypothesis: There exists multiple (three) distinct Calcium channels.
    Major results: Through analysis of different holding potentials, assessing the inactivation states of the different ionic currents, there were three Ca++ channels L/T/N type noted. These results were obtained through the electrophysiology technique of patch clamp and assessing cell activity.
    Overall conclusion: Three Ca++ channels exist that are in fact distinct from the sodium channels based on their specific components (e.g. potential, voltage dependence, inactivation states).

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    Authors opt to use a patch clamp in order to assess the different L/N/T type calcium channels which is well suited to study these various ion currents. Based on class discussion, the patch clamp serves as a robust technique in order to assess the activity of various ionic currents. The results from the various experiments allude that there are indeed different Calcium channels given the distinct holding potentials noted in Figure 2. The authors do not make notable mention of the controls used, however given the general hypothesis that different Ca++ channels exist, it could be proposed that their comparison to the Na+ channel was a control, which provided further confirmation that the channels were not sodium channels but instead calcium channels based on the voltage dependence. Thus the results are supported by the data provided.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    Authors provide modest introduction/discussion/conclusion sections. The authors’ rationale for investigating the existence of different calcium channels is thoughtful, stating the calcium channels are implicated in cardiac activity, which has major clinical relevance and warrants the need for further understanding of these distinct channels. Additionally the authors’ discussion on how they were able to determine the Ca++ channels from sodium channels demonstrates their understanding of the different voltage gated channels (e.g. by observing the voltage dependence). The authors results support the overall conclusion that there are multiple calcium channels, however their conclusion/results could be strengthened by including prior data from related studies done in the field that guided the studies they completed. Stated another way, the authors could have included more of the literature on calcium channels or lack thereof, in order to corroborate their hypotheses and subsequent findings.

  3. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    – Hypothesis: There is more than one type of Ca conductance in neurones
    – From whole-cell recordings under ionic conditions, three distinct components of Ca channel current were found; one current decays slowly and was given the label ‘L’ (long-lasting), another component decays rapidly and was given the label ‘T’ (transient), and the third shows qualities of both T and L and was given the label ‘N’ (neither T nor L)
    – The differences between the three channels include: slope conductance (L-type is the largest, followed by N-type, and then T-type), inactivation properties, and unitary current size
    – All three channel types have different responses to Bay K 8644 (a dihydropyridine Ca agonist); it enhances L-type currents in DRG neurones and does not change the T- or N-type channels

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    – There was mention of significance for some results, but there was no discussion of any statistical analysis
    – While there was some inclusion of methods, it was scattered throughout the paper and would be very difficult to reproduce their experiment, especially given that reagents that are mentioned are not specified in where they were obtained
    – By using a blocker and an agonist, it is possible to see differences in sensitivity and responsiveness between the three channel types, beyond just the differences in kinetics and unitary conductance

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    – Except for a few sentences in the abstract at the beginning, there is no introduction and no background into what the investigators are studying
    – The very little background in the abstract uses several sources, but does not elaborate on them or explain their significance beyond stating that “there is considerable evidence”
    – There is no rationale given; beyond a few sentences at the very end of the paper, there is no consideration of future directions or what these findings could mean in a more holistic setting
    – The paper does not discuss why the coexistence of three types of neuronal Ca channels with different sensitivities is important, how it contributes to the field, or how it relates to other literature beyond a brief mention of reports done by others

  4. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Hypothesis: This study did not have a specific hypothesis but was more exploratory in nature, with the authors investigating what types of neuronal calcium channels they might find in their model organism (8-12 day-old chick embryo), i.e. how the response to dihydropyridines (compounds used for labeling in Ca2+ channel purification) and electrophysiological properties might differ between Ca2+ channels.
    Results: Using electrophysiological methods, (i.e. whole-cell recordings, single channel recordings, voltage clamp), the authors identified three distinct types of Ca2+ channel in sensory neurons of chick dorsal root ganglion: N (activated by pronounced depolarizations and inactivated by extremely negative potentials), T (opened by weak depolarizations, contributes phasic current), and L (activated by strong depolarizations, provides current with long half-life). These three channel types also had distinct responses to Bay K 8644 (dihydropyridine Ca2+ agonist), with only L-type currents being enhanced by treatment with the compound.
    Conclusion: There are at least three different classes of Ca2+ channel in chick dorsal root ganglion neurons which contribute to calcium currents across the cell membrane, and these channels have differing sensitivity to the Ca2+ agonist used (Bay K 8644).

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    Electrophysiological techniques used were appropriate to the goals of the study, since methods such as voltage clamp enable researchers to examine aspects of channel activity, like current, that make distinction between channel types possible. Chick embryos are a well-established model organism. Necessary controls appear to have been used in experiments such as the one with Bay K 8644, where the authors recorded currents from all three Ca2+ channels before and after application of the compound. Not extensive statistical analysis, but authors appear to have performed calculations, i.e. for current relaxation, that are necessary for comprehension of the data. Data provided clearly illustrate unique currents associated with N-, L-, and T-type channels in the population of neurons studied, as well as a dramatic enhancement of L-type current (but not N-type or T-type) in the presence of Bay K 8644.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    Introduction: There wasn’t a true introduction in this paper, merely cursory introductory material presented at the very beginning. The authors briefly touch on the importance of determining the types of calcium channels found in neuronal membranes (key to understanding the diverse functions of Ca2+ within the neuron), and mention that little work has been done on single channel Ca2+ properties, thus providing justification for their study (it makes a contribution to basic knowledge). However, in-depth contextualization in relation to literature in the field was lacking.
    Discussion: Like the introduction, the discussion is brief and perfunctory by modern standards (only a couple of paragraphs), and is not clearly labelled. However, the authors do explain how their work makes a unique contribution to the field (“first report” to examine three distinct Ca2+ channels in a single cell type). The authors mention work done by other labs that supports their findings (i.e. single-channel recordings in ganglion neurons that indicate T- and L-type channel currents). The authors touch on the implications of their findings, speculating that the different Ca2+ channels could have specific purposes in the cell, but only in generic terms. In addition, little effort is made to explore the future directions in which the authors hope to take their research, though they do mention that it will be of interest to explore the types of cells in which these channels occur, how they are affected by different neuromodulators, etc.
    Conclusion: As a whole, the data presented convincingly demonstrates the presence of L, T, and N Ca2+ channels in chick dorsal root ganglion neurons, as well as their varying sensitivities to a dihydropyridine.

  5. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    -The authors hypothesize that there are multiple different types of calcium channels that exist in neurons that contribute to modulation of firing, neurotransmitter release, etc.

    – In Figure 1 of the results the authors show evidence of three distinct components of Ca++ channel currents in whole cells. They show that weak depolarizations evoke a long acting channel current. Another current becomes noticeable from -60 to -10 mV and decays rapidly. A third component of the calcium current appears with strong depolarizations. This third component is designated as “N” while the first two are designated as “L” and “T” respectively. In figure 2 the authors show that recordings on patches of DRG show three types of Ca channel current that is distinguished by different slope conductances. In the final two experiments the authors recorded from cells that had N-type channels but no T or L type channels. They also show that the three channel types differ in their responsiveness to a dihydropyridine Ca agonist. This drug strongly enhances L-type channel currents but not T or N types.

    – In conclusion the authors report that this is the first study to show the coexisitence of three Ca channel types in one preparation and that this may be translational to other whole-cell studies reported by others. They propose that these different channels may serve different functions (i.e. T-type being responsible rhythmic activity, and L and N being responsible for dendritic spikes or NT release.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    – The description of the methods is limited in the article, possibly due to journal restrictions. It would have been nice to see more experimental detail on the specific techniques used for their voltage-clamping techniques since they jump right into the results from the introduction.
    – For their controls in the third and fourth study it would have also been nice to see cells having only L or T type channels since they are looking at the function of cells which only contain N type cells. This type of control would have been a good comparison.
    – The data analysis is clear and straight forward and they did a good job at presenting their channel current averages in graph form which made it easy to observe for the reader.
    – In summary, their results are substantiated by the data provided in that they show that there are three distinct types of Ca channels in the cell preparations and that each component is distinguished by different conductances.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    – The introduction is very brief and does not provide much detail on the previous literature in the field. For example, they simply state there is considerable evidence for the presence of more than one type of Ca conductance, however they do not elaborate on what this evidence is. They also could have described this previous literature more in order to provide the scientific premise of the study.
    – Overall their results so support their conclusion in that there are three distinct type of Ca channels and they can be measured by their differences in conductance. They briefly speculate that all three Ca channel types may be found in whole-cell preparations from other studies in their conclusion as well, but they reference their own figure from this current study and do not explain how their findings of the coexistence in the preparation can be translated to other studies.

  6. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    H: There exists multiple types of Ca conductance in neurons and other cells.
    R: Using chicken DRGs, three different components of Ca channels were explored by applying various levels of depolarizing test pulses from different holding potentials. Different forces of depolarization and durations of depolarizations were observed. Concurrently, the authors further distinguished three types of unitary Ca channel current by recording cell-attached patches on DRG cell bodies. Multiple slope conductance sizes were observed as well as the inactivation of channels at varying levels of voltage. Furthermore, the authors focused primarily on N-type Ca channels without any L-type of T-type activity. Kinetic analysis of recordings from cell attached patches for various N-type Ca channels were analyzed. The authors recorded that as test pulse depolarization levels increased, the rate of change of current became steeper. Finally, application of a Ca channel agonist Bay K 8644, produced different single channel currents between channel types.
    C: The authors concluded that 3 different Ca channel subtypes existed (L, T, and N) and these channels have various characterizations to distinguish them. The kinetics of Ca channel subtypes are distinct and set these channels apart. Different subtypes are activated and inactivated at varying voltages and remain active for different durations. The rates at which Ca channels dynamically become activated and inactivated is also dependent upon channel type. Ca channels also respond differently to Ca channel agonists.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    M: Overall, the assays used to monitor the activity of the channels was pretty standard, using patch clamps to measure currents under various conditions and voltages. This method was pertinent to compare the duration of currents and strength of conductance necessary to identify multiple channel subtypes that could account for changes in these characteristics. While testing a channel agonist to show that it selectively affected the activity of L-type channels; comparing other agonists that had different selectivity or possibly using an antagonist (if available at the time) to show a reversal of these effects would further strengthen the notion that the effects of Bay K 8644 were L-type channel dependent. Controls were not very well used in their assays. Removal of any Ca from the environment to show a lack of similarity between currents would have further proven the third channel they identified was in fact a Ca dependent channel. Also measuring the effects of other electrolytes such as Na and K would have also further strengthened their proof that these channels, especially the newly identified N-type, were strictly mediated by Ca. Testing multiple cell types other than DRGs, I feel is important to support their original hypothesis.
    R: A lack of explanation for data analysis was clearly evident. Statistical significance for differences between channel subtypes identified was not provided. However, their rationalization of the results was clear enough evidence to support their conclusions. By comparing current duration and conductance size, they were able to argue that at least three channels were mediating the recorded differences in characteristics.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    I: While the authors mentioned that previous research supported their rationale for designing and executing their experiment, they did not go into very much detail. They lumped most of their background research into one cohesive idea supporting their hypothesis: there was prior research that supported their hypothesis. They did not go into detail on why this prior research was effective for rationalization nor why their approach was the best method for exploring their hypothesis.
    D/C: At the end of the article, the authors provide literature that supports their current findings such as previous findings in whole cells and single channel recordings of DRG neurons that showed kinetically different T-type and L-type Ca channels. They also further concluded that the different characteristics of Ca channels may be essential for different cellular functions specific to each channel subtype. I believe further in-vitro, in-vivo and ex-vivo work is necessary to come to this final conclusion.

  7. Donald Jessup

    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    Hypothesis – Multiple calcium channel subtypes are present in neurons, and specifically in the dorsal root ganglia neurons of chicks.

    Methods – Ion channel activity of chick dorsal root ganglia neurons was assessed in isolated tissue which was then incubated for 2-7 days. Taken in whole cell recordings 3 distinct components could be distinguished kinetically by applying various test pulses at different holding potentials. 1.) At holding potential of -40 mV strong depolarizations ( -10, + 10, +20 mV) were required to activate an inward current with a slow decay (t/12 of hundreds of milliseconds) and was designated the L-type or Long lasting type calcium current. at -60 mV a second current becomes visible, with a constant amplitude between -40 and -10 mV, indicating a very negative range of activation. This component decays rapidly ( r- 25 ms at -30 mV) and is so named T – for transient. A third component the current appears with strong depolarizations at -100 mV (Fig 1 a,c). The decay amplitude plateaus between -30 and -10 mV and grows substantially with subsequent test pulses, peaking at +20 mV. Termed ‘N’ (neither T nor L). Like T, but unlike L, N contributes phasic current and requires strongly negative holding potentials for complete removal of inactivation. 2.) Recordings from cell-attached patches on dorsal root ganglia cell bodies show three types of unitary Ca channel currents, distinguished by their different slope conductances in 110 mM Ba (Fig 2). These unitary recordings also demonstrated differential kinetics (opening, closing, inactivation) for each of the three type L, N, and T (Fig 2). 3.) Cell attached patches also demonstrated that N-Type channels open probability increased between -20 and +30 mV as well as showing a complete inactivation at a holding potential of -20mV, both of which support the whole cell data observed. 4.) The channels also differ in their response to experiments with the dihydropyridine Bay K 8644 (Ca agonist – elevates opening increasing Ca influx) showed that the compound would strongly enhance averaged L-type channel currents in DRG neurons (Fig. 4), but had no effect on T-type or N-type channels.

    Conclusion – Based on the differences in kinetics, thresholds of activation and response to dihydropyridine, the three components identified are the result of three distinct calcium channel species.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    I very much enjoyed the systematic approach the authors used in this paper starting a whole cell recording that showed several currents contributing to the net calcium current seen in figure 1 and then progressively investigating the identities of each one before switching to a different method of patching to look at single ion channels and pulling out their individual kinetics. It really shows the power of the technique and it’s unique ability to look at these fundamental properties that we can ultimately pharmacologically manipulate as seen in the experiment with Bay K 8644. The results are well substantiated by the simple but probative experiments performed and overall the authors are well within the given results to strongly conclude that the currents are coming from three different channel types.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    This paper was first published in 1985 and while the introduction does establish the rational for study very succinctly, it still feels too brief at least by today’s standards. It would have been nice to see a more substantial exploration of the relevant prior literature but the introduction still provides enough to follow the authors initial justifications for investigating these currents. The conclusion similarly is also brief, but does go into a few of the authors suppositions about the possible implications of their findings some of which would actually be proven correct (such as N-Type being related to vesicle release in synapses). Overall this paper cuts write to the results of their experiments and does not overstate or overemphasize their results in the conclusion. Perhaps the only thing that would have been nice to see is some in-vivo data demonstrating the persistence of these physiological observations outside of cell-culture data but it’s a very minor critique of a very thorough (if brief) manuscript.

  8. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    The authors hypothesized that multiple types of neuronal calcium channels existed in cells with different activities and sensitivities to modulators. The rationale for studying this topic was that understanding calcium entry would contribute to knowledge of different neuronal functions such as neurotransmitter release, rhythmic firing, and spike initiation. The dorsal root ganglion of the sensory neurons of chick embryos were used to test for the existence of three distinct Calcium channels. Depolarizing pulses were applied from a holding potential, and kinetic responses were recorded in order to distinguish the activities of each channel type. It was found that there was a channel that elicited a long-lasting current with a strong depolarization (L-type); another one was activated at weak depolarizations and had a transient current (T-type); and the third was unlike the previous two because they required strong negative potentials for removal of activation, and required a strong depolarization for activation (N-type). N-type channels were sensitive to the inorganic blocker Cadmium like L-type channels, but unlike T-type channels. Further, the three channel types differed in their response to the dihydropyridine Ca agonist, Bay K 8644. It enhanced L-type channel currents but did not affect T-type or N-type channels. The authors concluded from these experiments that at least three distinct types of calcium channels with different functions coexisted in the neurons.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    • Overall the methods were appropriate for this experiment. Patch clamp techniques were used and the data substantiated the results. Whole-cell and attached-cell patch clamping was used to characterize the channels using a Tyrode’s solution which included sodium channel inhibitors, which was appropriate for this study. The authors also distinguished between the three types of calcium channels by recording responses such as sensitivity for agonists and antagonists
    • I appreciated the inclusion of the three types of unitary calcium channel activity in the patch clamp recordings as it was easy to compare each.
    • A dihydropyridine agonist (Bay K 8644) was used on each channel to record its effects, and cadmium was used as a calcium channel antagonist. Additional agents, both agonists and antagonists, should have been used to confirm the found effects to make the data even more convincing. However, I appreciated the use of multiple techniques and methods to tease out the differences between the channel types.
    • The authors could have also looked at different cell types, such as cardiac cells, in order to determine if the calcium channels existed elsewhere in the body. One concern I had was how translatable cells from a chick embryo are to human nerve cells.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    There was no introduction with relevant background information or discussion around the relation of this study to current literature in the field. Only a small amount of discussion was present on the relevance of the findings; however, the speculation of the functioning of the different channel types was interesting: T-type channels could be contributing to threshold behavior or rhythmic activity, and N- or L-type channels could be contributing to dendritic spikes or neurotransmitter release. I would have liked to hear more on the relevancy and potential clinical implications; for example, calcium channel blockers with different targets could have different therapeutic effects in the body (L-type blockers for hypertension; T-type blockers for epilepsy).
    They mentioned that next steps are to determine the distribution of the different channel types on different cell types and their sensitivity to neuromodulators in order to determine their physiological roles. They should have included discussion on potential existence of subtypes of channels. Overall, the results support the conclusion.

  9. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    The hypothesis of this article is that the diversity of neuronal functions can be explained by the coexistence of different types of calcium channels. Whole-cell currents were measured using a cell-attached patch clamping technique. Test potentials ranging from -50mV to +20mV were applied starting from holding potentials -40mV and -100mV and the results showed three kinetically distinct profiles. Based on these profiles, holding and test potentials were selected for the next set of experiments which measured slope conductances that corresponded with the results obtained in the first set of experiments. Other experiments were carried out using cell attached patches containing only N-type calcium channels to further characterize this type of channel and contrast it to L- and T- type channels. The last experiment demonstrated different sensitivities of each channel to the DHPR agonist, Bay K 8644 and showed that it was selective towards L-type calcium channels. Overall, this study identified an L-type calcium channel which produces a long lasting current at strong depolarizations, a T-type calcium channel which produces a small current at weak depolarizations and an N-type calcium channel which requires strongly negative potentials to undo inactivation and strong depoloarizations for activation. It is noted in the conclusion that although this study was the first to specifically focus on characterizing distinct calcium channels, that evidence of their activity can also be seen in whole-cell current data from previous studies.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    The methods are clear and however it was written as a part of the caption to figure 1 when it probably should have been written independently. There are also details elsewhere that could have been included in the methods section. The whole-cell approach was appropriate for this study as there had been no previous reports of different types of calcium channels and so a single channel approach could not be taken initially. After obtaining the whole-cell currents and identifying the three types of calcium channels they could use a single cell approach and did so for their cell-attached patch experiment on cells containing only N-type calcium channels (single cell data was used to determine total number of channels). Tetrodotoxin was added to the external solution as a control to mitigate any signals from sodium channels interfering with the signals generated by calcium currents. Single channel currents from each of the three calcium channels were used as controls to show effect of Bay K 8644. Overall, the data and results are sufficient substantiate the conclusions of the study.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    There was a bolded abstract at the beginning of the article but I think there was a lack of an introduction. They briefly mentioned their rationale for the study but hardly expound on any of the previous findings in the literature that hint at the coexistence of individual types of calcium channels. The rationale is justified and the findings do relate to the literature in the field and they mention a few examples of where the findings of this study relate to other studies such as those focusing on dendritic spine formation or neurotransmitter release. Different channels should have different electrophysiological profiles and I think the experiments carried out in this study and the results obtained clearly support the overall conclusion.

  10. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Nowycky et.al. hypothesized the existence of a third type of calcium channel (N) and that a calcium channel-like agonist would increase the probability of the opening of L-type but not T-type or N-type calcium channels. The major result extracted was the defining identification by activity of the third type of calcium voltage channel which they define as N-type and the lack of activity that Bay K 8644 had to open on N and T-type calcium channels but observable activity in the opening of L-type channels. The overall conclusion is the identification of the N-type channels; N-type channels differ from L-type channels because they need stronger negative potentials (about -80 mv) to begin its opening and N-type channels differ from T-type channels because they need stronger depolarizations for activation to fire (about +20 mV).

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    Figure 1 – I liked the fact that Nowycky et.al laid out details of their methods and the components of them such as where the cells came from (DRG from 8-12 day old chick embryos) although they did not state the reasoning for that the use from that specific animal/age. I liked that they mentioned isolation and culturing of the cells and the living conditions of the cells (describing the media they were cultured in before recording began). What I thought was great (about all the figures) is that they describe the components of the inside of the pipette (that will break through the cell) as well as the external solution where the cell needs to be placed. For this figure I would’ve have liked to see a control (other than L and T-type channels) although I’m not sure if there would be any relevance by having done that? I get that maybe both L and T-type channel activity was known so that might’ve served as the control, but it is always nice to see a different system. The results from this figure were easy to follow in order to make the comparison of the differences.
    Figure 2 – For these cell-patch recordings Nowycky et.al actually mention different cell types used per channel type which I thought was interesting to do. Why not use the same cells per channel? At the bottom of the description they state that tons of combinations have been possible but probably too many to show – but it would’ve still been nice to see the same cell line per channel and what they showed.
    Figure 3 – this did a good job to focus on the activity of the N-type channel alone by describing its overall test potential and holding potential and its depolarization activity. Panel b was a great representation to show the probability of voltage-dependence of peak channel opening.
    Figure 4 – This result was the most straight forward of them all. Firstly, Nowycky et.al showed the results of a control which drove the message perfectly. The result here is that this agonist, Bay K 8644 strongly increases the opening probability of only the L-type channel but not the others. One slight thing extra that would have been nice was to see different concentrations of the drug tested.
    Overall cons were the lack of statistical analysis shown per comparison.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    This article had an interesting introduction – it was basically the abstract and the introduction. It did the job of relaying the significance to investigate their question, so it is not an overall huge deal. Although I feel like in today’s standards Nowycky et.al would’ve had to explain deeper as to why the calcium channels were important to study geared towards understanding human disease (maybe addiction)? Again, this article does not have specific discussion/conclusion segments to it but Nowycky et.al do a good job to go through their conclusion per result. Their light discussion section touches on the primary bases (the co-existence of all three channels), the unitary activity of L and T-type channels, and the role that N-type channels have (specifically how N-type compares to L and T-type). Throughout the article Nowycky et.al do a good job at detailing specific citations for further clarification, which I really liked.

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