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  1. Critique Student Aminatta Tejan-Kamara Instructor Dr. Nicholson Date 10/23/19
    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: Chronic stress will induce anhedonia and alter glutamate release and BDNF trafficking.
    Major results:
    • dysfunction in glutamate/GABA release is in part restored through subanesthetic dose of ketamine- transmitters were measured and cross compared in control group, CMS-resilient group and CMS-vulnerable group.
    • impairment in the dendritic BNDF trafficking caused by CMS is fully restored by subanesthetic dose of ketamine- the authors implemented the in situ hybridization to analyze changes
    • CMS causes major phenotypic changes, the authors implemented the CMS model with the behavioral assay SPT two bottle paradigm.
    Overall conclusion is that CMS causes changes in Glutamate release and BDNF trafficking in mice vulnerable to stress and ketamine offers some improvement in these impairments/changes.

    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 opted to implement the chronic mild stress model in order to simulate environmental stress component in MDD, the model according to prior literature is said to be a reasonable one for stress simulation, but there have been questions raised about the reliability/replicability of the model and subsequent data, experimenters could have attempted to complete follow up studies that demonstrate the results are in fact replicable. Regarding the SPT two bottle paradigm for anhedonia, the 55% cutoff to assess anhedonia seemed arbitrary. Given the rat body weights varied by 15 grams, it is possible the experimenters could have assessed the weight percentiles among/within their groups and assign anhedonia in this manner as opposed to the general proportion of 0.55. Additionally, the route of administration for ketamine was I.P. likely for the fast onset to get drug on board, however, human studies administered S.Q. injections, experimenters could have attempted to administer ketamine subcutaneously and analyze data that may have more translational value. Overall the experimenters study design is reasonable, with no major concerns. The figures, particularly the HPA axis in conjunction with body weight gain are compelling for the experimenters 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.

    Introduction provides reasonable background that leads well into the rationale behind the study. The authors offer a decent literature review in the discussion and do well to acknowledge the novelty in their findings and how the work will add to the knowledge in the field. Results do support overall conclusion that chronic stress in vulnerable rats causes changes in glutamate release (more inhibition of glutamate) and that ketamine has some restorative action, these findings will allow for translational efforts in finding better therapeutic treatment for individuals with treatment resistant MDD.

  2. In stress-vulnerable rats, the Chronic Mild Stress model of depression induces anhedonic behavior, impairment of glutamate/GABA presynaptic release, impairment of BDNF mRNA trafficking in dendrites, and impairment of dendritic morphology in the hippocampus. The purpose of this study was to determine ketamine’s restorative properties and potential in treatment of depression and chronic stress. In this study, 144 male rats were exposed to stressors once or twice daily for five weeks. The rats were also habituated to sucrose and this was tested via several sucrose habituation tests. The rats were given either saline or ketamine IP 24h prior to sacrifice. Weight gain and serum steroid levels were also monitored. Synaptosomes were purified and exposed to KCl and were then analyzed for endogenous glutamate and GABA by HPLC. BDNF protein levels were measured from HPC via ELISA. Digoxigenin (DIG)-labeled riboprobes were utilized to measure BDNF, BDNF-2, and BDNF-6 transcripts. In situ hybridization was then performed on coronal slices after sacrifice. Maximal distance of hybridization signal in dendrites was measured. Also immediately after sacrifice, left or right hemispheres were processed for Golgi staining. Dendrites were reconstructed for analysis and dendrite length, dendrite branching, and Sholl analysis were assessed. The SPT found that ketamine administration resulted in a significant increase in sucrose preference but only in CMS vulnerable rats. More testing showed no significant changes in resilient rats. Furthermore, CMS rats showed a decrease in body weight compared to control rats and weight of adrenal glands and serum cortisol levels were also markedly higher in CMS-V than CMS-R and both were higher than in CNT rats. Ketamine had no effect on these three measures. With regards to glutamate release, ketamine only seemed to affect basal release in CMS-V, with a strong trend toward normalization. With regards to GABA release, CMS reduced the release of GABA in CMS-V compared to CNT, which was able to be restored by ketamine. Total BDNF mRNA levels were significantly reduced by CMS in both CMS-V and CMS-R, and KET treatment had no effect. Ketamine’s effect did not discriminate against different BDNF variants expressed in rat brain. In situ hybridization studies assessing changes in BDNF mRNA dendritic localization found decreased trafficking in both CMS-R and CMS-V rats and ketamine had restorative properties in the CMS-V population. Ketamine showed reversal of reduction in trafficking of BDNF variants 2 and 6. Furthermore, CMS significantly reduced the total length of apical dendrites in the CA3 of CMS-V and ketamine had restorative effects on this as well.
    I appreciated that all rats were coded and analyzed blindly in the in situ hybridization experiment. One question I had was how exactly they determined that differences in sucrose preference was a result of stress vulnerability and was not due to preexisting differences in preference for sucrose. I was also wondering why sucrose preference is an accurate method of assessing anhedonic behavior and depression? Wouldn’t it have been more indicative of anxiety or stress rather than depression? What are some other alternatives that might’ve been used and why was sucrose chosen over them? They also didn’t use a control for testing ketamine in the SPT. They should’ve had a group of rats that were not exposed to any stress and then given ketamine to look for any changes due solely to ketamine even when not in the presence of a stressful environment. Their methods of statistic analysis seemed fairly appropriate. Overall, it seemed their results did substantiate their claims for the most part but it seemed there were some components of the study that could’ve been better carried out.
    Overall, the introduction was very clear and brief and the paper itself was well-written. The discussion still left several questions unanswered, especially regarding how exactly ketamine influences BDNF trafficking, so while the paper offers a nice overview on some of the effects that ketamine has in stress-vulnerable rats, it still leaves a lot of details unanswered. They did address numerous other studies so their background research was thorough. With regards to future study, it would be interesting to see if continued ketamine administration either led to a build up of tolerance and decrease in therapeutic effects or if ketamine reduced recurrence of maladaptive changes seen as a result of stress. It also would be interesting to see whether differences in how chronic the stress is affects the restorative properties of ketamine. For example, would ketamine have a more profound effect on rats exposed to stress for 30 days compared to rats exposed to stress for 30 weeks?

  3. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Tornese et al. hypothesized that a single dose administration of racemic ketamine has the rapid ability to restore the side effects produced by chronic mild stress (CMS) in a rodent model. The major results are that ketamine is able to quickly restore anhedonia-like effects in vulnerable rats (see fig. 1D), ketamine is (partially) able to restore presynaptic decreased levels of glutamate and GABA in vulnerable rats (see fig. 2), and that ketamine was not viable in restoring expression of BDNF mRNA and protein levels in the hippocampus (see fig. 3). The overall conclusion of this article is that acutely treating rodents with a single dose of ketamine was able to restore behavior and cellular alterations produced by different assays which mimic depression-like behavior.

    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.
    – Method/result fig.1: the methods applied to the results presented in figure one was the introduction of their experimental plan which I thought was designed fairly for the overall results quantified. They had good habituation time, although I would’ve probably allowed the rats to habituate more to the sucrose preference test (SPT) for more than one day prior to the start of the CMS (to get a better overall baseline). Although Tornese et al. describe the types of stressors rats were exposed to I thought that in the results they could’ve have expanded more as to which type of specific stressor was being quantified (I guess I don’t agree with the fact that all 8 stressors were compressed into one CMS). I do like the fact that they showed the weight changes throughout the experiential procedure. Statistics ran in this section included good comparisons and good use of control between an actually control (CNT) and control within the groups CMS-R, CMS-V and CMS-V+KET.
    – Method/result fig.2: this is based on their major conclusion that ketamine is able to partially restore glutamate and GABA in the hippocampus. Although I am not sure that I agree with this conclusion, even though they do use the word “partially.” In this section Tornese et al. are comparing the CMS rats basal release of neurotransmitter (a,c) and depolarization-evoked neurotransmitter levels (b,d). In a (glutamate) they are basing their conclusions of a result that is not shown or done – comparing CMS-V+KET and CMS-V there was no increase in glutamate level and in c (GABA) their conclusion is based off a result that does not show a significant increase for the comparison made CMS-V+KET to CMS-R (which is the wrong comparison they are making. They should’ve stayed consistent and compared CMS-V+KET to CMS-V).
    – Method/result fig.5: In this section Tornese et al. are showing the effects of ketamine on apical/basal dendritic re-growth through dendritic analysis of CA1/CA3 areas in the hippocampus and Sholl analysis. There is more significance in apical length/number of branches restored after ketamine and no significance in changes of basal dendrites (which I find interesting) and Sholl analysis shows the same effect.
    – I think that the best consistent statistics and controls described are in figure 1, Tornese et al. don’t really keep consistency throughout the article. Figure 1 compared to the ones described and ones not described had the best overall result/conclusion pair. They did describe more methods such as qPCR, ELISA, and in situ hybridization.

    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.
    Tornese et al. have a thorough introduction of what the main clinical goal this work has – MDD. They describe the symptoms, dysfunctions, and the underlying hypothesis for using a scheduled drug to help treat MDD. They do a good job at citing many sources in the field to aid the issue. They have a good scientific premise behind their rationale for this work although I do think that their conclusions are a bit stretched out for the data presented. Their discussion was a bit offsetting because they introduce more data after the short discussion which I thought was weird. Also, as they do conclude many things throughout the article their final conclusion was very short and re-state the findings without emphasizing on what direction this work could go to next. For instance, testing different doses of ketamine, testing its enantiomers (R/S) to see if their effects are different for the already described experimental plan.

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

    Hypothesis: Though the hypothesis is not directly stated, it is implied to be that effects of chronic mild stress (a model of major depressive disorder) on glutamate system dysregulation and anhedonia in male Sprague-Dawley rats are only observed in stress-vulnerable animals, and that these effects can be mitigated by ketamine administration. Using behavioral (i.e. models of chronic mild stress) and biochemical (i.e. qPCR, ELISA) assays, the authors determined that certain rats were vulnerable to chronic stress effects, including anhedonia, impaired glutamate release, and reduced dendritic BDNF mRNA trafficking. These effects were at least partially reversed by a single administration of sub-anesthetic ketamine. Based on these results, the authors concluded that ketamine may exert its antidepressant effects by re-establishing normal dendritic BDNF and glutamate/GABA release in individuals susceptible to chronic stress.

    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.

    Methods: Chronic mild stress is well-established in the literature as a model of major depression in rodents, and several of the stressors (i.e. soiled cage) are consistent with versions used by other labs. However, authors could have been clearer regarding certain stressors, i.e. What did they consider “overcrowding?” When they say that food and water deprivation happened for “up to” 12 hours, was the deprivation period consistently 12 hours long? A little more detail here would have been helpful in the interest of enhancing reproducibility. Sucrose preference test is a standard measure of anhedonia, making it a logical choice for assessing whether the authors’ CMS model induced depression-like symptoms in their rodents. However, I was confused about the way they ran SPT; were the animals housed in individual cages during these test periods (Animals section mentions they were normally pair-housed)? Otherwise, it would be extremely difficult to determine how much sucrose solution each rat drank. Measurement of plasma CORT is an established marker for the effects of physiological stressors. Golgi staining and dendritic analysis were both useful means of determining changes in neuronal structure due to CMS, ketamine treatment, etc. qPCR, ELISA, and in situ hybridization all useful techniques to assess measures like BDNF levels. I liked that the authors used several different methods across a range of disciplines (from behavioral to biochemical) in order to get at the potential mechanism of ketamine’s antidepressant effects. I was curious to know why the authors chose 55% sucrose preference as the cutoff, even with the literature citation. Should they have used a slightly higher cutoff to ensure that CMS-R rats were truly resilient? Also, why were animals sacrificed so soon after ketamine administration before longer-term behavioral effects could be studied. Would have liked to see data for females (incidence of depression quite high among women). Some inconsistency with sample sizes (i.e. animal number for in-situ hybridization was smaller than other experiments) Controls: In general, appropriate controls seem to have been used. Data analysis: One-way/two-way ANOVA followed by Tukey’s appropriate test for differences between multiple groups; unpaired two-tailed Student t-test is appropriate for comparing differences between two groups, etc. Results: Generally support conclusions, though flaws in experimental design should be kept in mind.

    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: The introduction clearly describes what is known about the link between major depressive disorder and chronic stress/glutamate dysregulation, as well as the previous literature on ketamine’s apparent antidepressant action. The authors emphasize the need for greater knowledge of ketamine’s mechanism of action (BDNF may potentially play a role, but it was unknown at the time of the study whether BDNF mRNA trafficking was affected by ketamine treatment or chronic stress). Solid rationale for present study.
    Discussion: Some context provided for how results fit into previous studies on depression/glutamate, discussion of potentially novel results (i.e. CMS effects on presynaptic glutamate release might only be seen in stress-vulnerable rats). Overall conclusion is supported by data from stress-vulnerable rats.

  5. Based on the understanding of the glutamate/GABA system and the involvement of ketamine of an antagonist of the NMDA receptor, the authors hypothesized that ketamine would be able to ameliorate symptoms of depression such as anhedonia, and related phenotypes such as reduced dendritic trafficking and atrophy as well as levels of Brain-Derived Neurotrophic Factor (BDNF). It was found that ketamine affected hedonic behavior subjected to chronic mild stress (CMS) by first performing sucrose preference tests to deem them resilient or vulnerable to mild stress. In CMS vulnerable rats there was decreased weight gain. CMS reduced presynaptic release of glutamate and GABA in the hippocampus of rats, and ketamine administration restored these levels. Further, CMS was shown to cause reduction in expression of BDNF mRNA in the hippocampus, and KET administration did not restore this effect; however KET did restore levels of dendritic trafficking of total BDNF mRNA and BDNF splice variants. Finally, ketamine restored the retraction of CA3 apical dendrites in the rat hippocampus. Therefore the authors concluded that ketamine administration in stressed and vulnerable rats reversed anhedonic behavior as well as partially restored glutamate and GABA release, and completely restored BDNF dendritic trafficking and dendritic atrophy.

    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.
    • Only male mice were used, which is a significant limitation as depression often manifests different in males and females.
    • They used a clinical model of chronic stress mice which seems like a strong model, but it may be hard to reproduce and keep consistent. Multiple stressors were applied to the rats including shocks and forced swimming. This would be hard to keep consistent.
    • Statistical analysis was appropriate; one way ANOVA with a Tukey’s post-hoc test, t-tests, and two-way ANOVAs were used depending on the experiment. Also, appropriate controls were used.
    • There was a cutoff of 55% for showing sucrose presence and then deeming them as resilient or vulnerable- this seems like an arbitrary cutoff and doesn’t convince me that the preference is strong or distinct between groups
    • Multiple splice variants of BDNF were analyzed which is a strength of the paper.

    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 does an excellent job in establishing the rationale for the paper; it discusses the fact that major depressive disorder is a debilitating disease that causes anhedonia, and that dysfunctions in the glutamate system have a primary role in this along with altered volume and connectivity in the corticolimbic regions of the brain. Further, since Ketamine had been shown to have antidepressant effects as an NMDA antagonist, which increases glutamate transmission, activating AMPA receptors and leading to the release of BDNF, the authors wanted to use this knowledge to see if it would restore behavior and dendritic morphology in CMS rats. One surprising finding was that KET did not restore reduction in BDNF mRNA and protein; the discussion went over this finding and explored the potential reasoning. The paper overall showed significant findings in which the results supported the conclusion. They discussed potential as use for an antidepressant but I would have been interested in potential adverse effects caused by chronic ketamine use. The conclusion was specific and sufficient in summing up the data presented.

  6. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Dysfunction of glutamatergic transmission in corticolimbic areas plays a key role in depression and combined with environmental factors such as stress, determines individual susceptibility to the disorder. Ketamine at subanesthetic doses has an antidepressant effect and is hypothesized to work through a mechanism involving re-equilibration of glutamatergic and GABAergic transmission and dendritic BDNF. A chronic mild stress (CMS) model was used to induce depressive, anhedonic behavior in mice which was quantified using a sucrose preference test (SPT). Weight gain, adrenal weight, and serum CORT levels were used as measure of stress. qtPCR and ELISA were used to quantify BDNF mRNA and protein, respectively and in situ hybridization was used to visualize BDNF in CA1 and CA3 regions of hippocampus. Ketamine was shown to restore anhedonic behavior in CMS vulnerable mice in SPT and increase presynaptic release of glutamate and GABA in hippocampal neurons. Ketamine had no effect on BDNF transcription or total protein but restored dendritic trafficking of BDNF in CA3 apical hippocampal dendrites. Overall it was concluded that there is a relationship between vulnerability to chronic stress, glutamate/GABA release and BDNF availability and that ketamine restores the homeostasis of these factors to elicit its antidepressant effects.
    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.
    Sprague-Dawley rats were used in this study and I think it was appropriate that they used an outbred line because it was important to show that there is a genetic component which imparts vulnerability or resistance to CMS treatment. I think the results showed that the experimental model is reliable; CMS treatment does produce changes in SPT and changes in physiological measures such as weight, adrenal gland weight, and serum CORT. In the interest of translatability however, I feel they could have had an additional behavioral metric to assess anhedonic behavior. Locomotion (open field test) or marble burying for example, could have been used as additional measures. In addition to anhedonia, depression manifests as reduced involvement in mundane activities such as one’s usual housekeeping or work duties. The control animals were not subjected to CMS and were not given drug at any time which is fine for the most part. The only problem I see with the control is that they did not measure SPT in rats that were not subjected to CMS (like control) but also received ketamine. Without such a control you cannot rule out the possibility that ketamine increases sucrose preference in non-CMS rats. If SPT scores were the same in control rats treated with vehicle or ketamine, then it could be concluded that ketamine induced restoration of SPT scores in CMS-v rats was due to the antidepressant effects of ketamine. The methods for investigating BDNF were appropriate; they looked at both mRNA and total protein using qPCR and ELISA, respectively and did in situ hybridization to corroborate findings in the field in regard to neuroanatomical changes in corticolimbic areas implicated in depression. The statistical analyses were logical, CMS-v values were compared against control and CMS-r rats and CMS-v-KET values were compared against CMS-v but they didn’t compare CMS-v-KET rats against control in all their figures. Lastly it would have been interesting to see how the antidepressant effects of ketamine hold up after repeated administration over the course of CMS treatment but overall I think the data substantiates the conclusions made in this 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.
    From the introduction, the rationale behind the study is quite clear; dysfunction of glutamatergic transmission is implicated in MDD and ketamine, an NMDAR antagonist, must elicit its antidepressant effects through modulation of the glutamate system. The discussion is sound and notes what conclusions cannot be made by the findings of the study. For example they note that although the antidepressant mechanism of action of ketamine involves enhanced glutamate release and BDNF translation but it is unclear whether the two events are ordered (one change induces the other) or independent of each other. Ketamine acts presynaptically at NMDA and GABA receptors to alter release of GABA and glutamate and postsynaptically at NMDA receptors to enhance BDNF expression and so the precise mechanism by which ketamine restores homeostatic plasticity is unknown. Overall the results were in accord with previous findings in the field and the conclusions that could be made from this study were fair. The genetic component to CMS vulnerability I feel is noteworthy and the authors failed to cite any literature with regards to genetic predisposition to depression that would have been relevant.

  7. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    – The hypothesis is that the Chronic Mild Stress (CMS) model of depression in rats produces changes in glutamate/ GABA presynaptic release, BDNF mRNA trafficking in dendrites and dendrite morphology in hippocampus along with anhedonic behavior in rats and that these biochemical and behavioral changes in vulnerable rats can be restored/ rescued by ketamine.
    – The major results are that CMS produces differing anhedonic behavior in rats (some are resilient, and some are vulnerable). They produced this behavior by putting the rats through chronic mild stressors such as food and water deprivation, isolation housing, light cycle reversal, forced swim, etc. They then tested ketamine in the vulnerable rat group and found that it rapidly reverses the depressed-like behavior. Next the authors show that the CMS produces phenotypic changes in vulnerable rats. This includes changes in body weight gain and increases in adrenal gland weight (vulnerable group only). CORT levels were also increased in the CMS groups compared to controls. In these three measures the vulnerable group had a greater change than the resilient groups. Next the authors measured glutamate and GABA release and transmission in the hippocampus. They found that presynaptic glutamate release is stable in resilient rats but lowered in the vulnerable rats and this was not restored by ketamine. Also, the GABA release measured was increased in the resilient group compared to controls. CMS also reduced release of GABA in vulnerable group, which ketamine fully restored to control levels. CMS also reduced the expression of BDNF mRNA and protein in hippocampus of both stressed groups. Ketamine had no effect on this expression. However, the reduced dendritic trafficking of BDNF and mRNA in vulnerable rats was restored by ketamine. In addition, ketamine reversed the shortening of apical dendritic spines in vulnerable rats. Again, this was tested only in the hippocampus of the rats.
    – In summary the authors conclude that the observed changes that are exclusive to vulnerable rats are partially restored by ketamine. They conclude that the anhedonic behavior as well as the GABA and glutamate release impairment is partially restored by ketamine. Also, that the impairment in dendritic trafficking is reversed by ketamine as well.

    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.
    – In the methods the authors used both a behavioral and ex-vivo approach. For their CMS model they cite a well cited paper, however many of the stressors they listed they used are acute stressors and may not be the most translational model of depression. For example, 12 h food deprivation in rats will certainly cause mild stress, however, if this is like chronic depression in humans remains questionable. In addition, they only administered ketamine to vulnerable rat groups, while they did not administer ketamine to the control or resilient groups. It would have been nice to see this control as well to determine any changes ketamine may cause on its own. Their data analysis is clear and significant effects are clearly labeled on the graphs. Overall their results are substantiated by the data provided in that they show phenotypic changes in the vulnerable CMS rat groups and that these changes are reversed by ketamine treatment.

    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.
    – In the introduction they give a good background on MDD and related dysfunctions in the glutamate system. They do discuss that ketamine has been shown to reverse preclinical and clinical signs of depression lasting several weeks after treatment. They nicely make their scientific premise for this with their CMS model design. In their discussion it seems they over-interpret a lot of their findings. For example, they write in the section 4.1 title that impairment in glutamate and GABA release is partially restored by ketamine. This seems a bit misleading in that the partial restoration was a full restoration in the GABA release but no effect on glutamate release. Overall, their conclusions seem to be selectively wording the results to make it fit more with their original hypothesis.

  8. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    H: It was hard to find an overall arching hypothesis clearly stated but considering the goal of the paper, I believe they hypothesized that trafficking of BDNF mRNA is regulated by stress and KET treatment.
    R: The authors showed that in stress-vulnerable rats, depression through a CMS model, induced depressed like behavior compared to resilient rats. They also showed that the same CMS model impaired glutamate/GABA presynaptic release. They showed that the CMS model induces BDNF mRNA trafficking in dendrites and dendritic morphology in the hippocampus. Finally, they showed results that single admin. of Ketamine restores normal behavior and a majority of cellular/molecular changes in vulnerable rats but did not restore BDNF mRNA levels.
    C: The authors concluded that the mechanism of ketamine works through re-equilibration of glutamate/GABA release, dendritic BDNF and by reversing synaptic and circuitry impairment.

    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: The authors used a baseline (pre-experimental) sucrose preference test to account for preexisting differences in preference for sucrose during their categorization of resilient or vulnerable rats. There are multiple ways to test stress vulnerability behaviorally in rats and I found it interesting that they used a simple sucrose preference test to conclude vulnerability. While adrenal gland weight, weight gain and CORT serum levels were provided, multiple mechanisms beyond stress could account for these changes. They also reported a reduction of expression of total BDNF mRNA and BDNF splice variants for both resilient and vulnerable rats which makes me question the level of vulnerability of the CMS-V group. The authors also failed to use a critical control when testing Ketamine: a vehicle treated control group. Finally, it would have been nice to see the effects of Ketamine compared to the current first-line treatments for depression such as an SSRI as a positive control.
    R: Proper statistical analysis along with post-hoc tests were reported for all data provided to ensure accuracy of the results. Although in Fig. 1B they show a significant difference in the CMS group in sucrose preference; how clinically relevant is a decrease of 20% sucrose intake to show that they induced CMS. They also showed that KET reversed the sucrose preference for CMS-V but it did not fully reverse the sucrose preference relative to CNT. They also didn’t show any effects of Ketamine for Fig.1 in the CMS-R group to compare between the CMS-V group. Their results for Fig. 3 and 4 while significant, did not seem clinically relevant at the changes of BDNF they were reporting.

    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: Although a lot of information was provided as background for the experiment; it became hard to follow their rationale as to why they chose to focus on the particular part of the mechanism. They explained multiple mechanisms that have been proposed over the years and explained many of them instead of explaining why they chose to focus on a specific mechanism.
    D/C: Overall, I think some of the results were stretched to support their overall conclusion. The vulnerability of the rats under the CMS-V group is questionable as far as clinical relatability is concerned. Furthermore, Ketamine only reversed some of the characteristics of the rats in the CMS-V group and only partially for others leading me to believe that another underlying mechanism is responsible for these CMS-V characteristics. The lack of vehicle controls also prevents me from making any conclusions regarding the effects of Ketamine.

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

    This study aimed to investigate the effects of stress and ketamine treatment on BDNF regulation. They proposed this research question based on literature suggesting that rapid activation of neurotrophic signaling and synaptic translation of Brain-Derived Neurotrophic Factor (BDNF) has been linked to the behavioral effects of ketamine. They used a Chronic Mild Stress (CMS) model of depression and showed anhedonic behavior in their Sprague-Dawley rats, and that glutamate presynaptic release as well as BDNF mRNA dendritic trafficking and dendritic morphology were impaired in this model. In addition, they demonstrated that single administration of sub-anesthetic ketamine was able to restore behavior and specific cellular/molecular alterations in vulnerable rats.

    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 methods such as: subjecting rats to the chronic mild stress model to induce depressive-like behaviors (n=96), sucrose preference test to assess the development of anhedonia-like behavior after induction of the chronic mild stress test, qPCR and ELISA to measure BDNF levels as well as in situ hybridization studies to assess changes in BDNF mRNA dendritic localization. They also measured phenotypic changes like body weight, adrenal/total body weight ratio and serum corticosterone levels over the course of their experiments. The methodological approaches used in this study were for the most part sufficient for the kind of proposed research question to be answered. However, I think they could have expanded more on their measures of anhedonic-like behaviors. They could have performed a battery of tests to establish anhedonia instead of relying only on the sucrose preference test. Other tests they could have included like the elevated plus maze test and even light-dark box test for anxiety-like behaviors. Recently, wheel running has been gaining traction as a measure of anhedonia and even the splash test could be sufficient to assess depressive-like behaviors. Overall, I think they could have done more to establish the development of anhedonia in the model. In addition, they subjected the rats to 5 minutes of forced swimming as a “stressor”; instead, the forced swim test could have acted as another measure of anhedonic-like behavior.

    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 does a great job at explaining the rational and premise of the study. The introduction does a sufficient job at giving adequate background and stating the problem state. They subsequently go on to express what their hypothesis is and to evaluate it using numerous approaches. The organization and flow of the article in terms of the methodology and the progression of the results and discussion were thorough. However, I am not convinced that all their conclusions are strongly supported by their results. For example, in section 3.4, they claim that chronic mild stress reduces the expression of BDNF mRNA and protein in the hippocampus; however, their data does not support this. In Fig 3b, BDNF protein level of CMS-V (with or without KET) actually showed no difference compared to controls, which they don’t address in the discussion. Pertinent to their conclusions, they claim that ketamine rapidly reverses anhedonic behavior in rats vulnerable to CMS paralleled by partial restoration of glutamate/GABA release in the HPC and complete restoration of BDNF dendritic trafficking and of apical dendrites atrophy. However, they do not include validation studies for this proposed conclusion maybe by experimentally atrophying BDNF mRNA at synapses in vivo by some other means and evaluating if that causes anhedonic-like behaviors in the rats and if it was reversed by ketamine treatment.
    Another comment I have is on the chronic mild stress model, some of the stressors do not seem to be considered mild. An example would be withholding food or water for up to 12 hours… which seems to me more like a severe stressor rather than a mild stressor. Lastly, the 55% cut-off preference rate for rats in the sucrose preference test seems very arbitrary although it has been published elsewhere, they do not state the rationale for using the certain cutoff in categorizing resilient versus vulnerable rats.

  10. Donald Jessup

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

    Hypothesis – The impairment of glutamate presynaptic release, BDNF mRNA dendritic trafficking and subsequent dendritic morphology in HPC is restricted to stress-vulnerable animals and can be recovered by a single low dose of Ketamine.

    Methods / Results – 1.) The first set of experiments used a Sucrose preference test was used to categorize rats as resilient or vulnerable to chronic mild stress, and was able to demonstrated that Ketamine rapidly restores anhedonic behavior in vulnerable rats. This was shown when saline-treated CMS-V rats still showed anhedonic behavior, while sucrose preference was significantly increased in CMS-V + ketamine. 2.) Chronic mild stress induced major phenotypic changes in vulnerable rats as demonstrated by significant decreases in body weight and increases in the adrenal gland weight in both CMS-V and CMS-R groups. Ketamine treatment did not reverse this effect. 3.) Chronic mild stress reduced presynaptic release of glutamate and GABA
    In the hippocampus of vulnerable rats shown by CMS significantly reducing basal glutamate release from superfused synaptosomes selectively in CMS-V animals ketamine affected only basal release in CMS-V trending toward normalization. 4.) compared to both CNT and CMS-R the total BDNF mRNA levels in whole HPC being significantly reduced in both CMS-V and CMS-R. Splice variants mRNA levels were also measured and it was found that BDNF-2,4,6 were reduced in all groups while BDNF-1 was reduced in all but CMS-R. Ketamine was again ketamine did not restore these changes. 5.) Chronic mild stress reduced the dendritic trafficking of total BDNF mRNA and BDNF splice variants in hippocampus of vulnerable rats. A significant decrease in the trafficking of total BDNF mRNA was found in the CA1 region of HPC of both CMS-R and CMS-V, with CMS-V showing the greater reduction which was recoverable by ketamine administration. CA3 only showed mRNA reductions in the CMS-V group which was likewise recoverable with Ketamine. 6.) lastly Scholl analysis revealed that chronic mild stress induced retractions of CA3 apical dendrites in
    hippocampus of vulnerable rats. This was evident in the reduction of the total number of branches of CA3 apical dendrites particularly in the CMS-V group and was ultimately recovered by ketamine treatment.

    Conclusion: Ketamine rapidly reverses anhedonic behavior in rats vulnerable to CMS, as well as partial restoration of glutamate/GABA release in the HPC, the complete restoration of BDNF dendritic trafficking, and the reversal of apical dendritic atrophy.

    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 paper was very interesting from a conceptual standpoint I don’t really have a problem with how they approached their questions and which experimental measures they utilized in the study. The only minor criticism I would make regarding methodology is the glutamate release synaptosome experiments would have been better supported with some very basic electrophysiology showing decreased synaptic strength or even reduction in field potentials. Particularly because they were examining the hippocampus which is perhaps one of, if not the most electrically accessible regions to do very simple physiological recordings. Overall the results are supported by the data that was presented and collected, and controls were appropriate and allowed for the proposed conclusions.

    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.

    Overall the introduction was very good, and established the prior literature well and provided the authors reasoning for pursuing this study while highlighting the novelty / knowledge gap that was being served by this study. The discussion is exhaustive and detailed, and was very good at both establishing what was known prior and how the results of this study may fit into the larger mechanisms currently being proposed / explored by this field. In particular the discussion really focuses on alternative interpretations of the data and that’s a feature that can often be either too short or not present in many papers and I really enjoyed reading through the alternative hypothesis and how that might relate to the underlying mechanism of ketamine’s rapid action of MDD. The only outstanding point of critique I found with the conclusion was, that the authors mention several times the AMPA activation prior to BDNF release and yet both the study design, and subsequently offered alternative explanations for mechanisms of action hardly mention the role of AMPA in this pathway. It would have been nice obviously if they weren’t going to look at AMPA, if they would have at least dedicated more of the discussion to addressing it.

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