Saltar a PREreview

PREreview del Incubation of oxycodone craving is associated with CP-AMPAR upregulation in D1 and D2 receptor-expressing medium spiny neurons in nucleus accumbens core and shell

por Cristina Marie Rivera Quiles y 1 otro/a autor/a del HHMI Transparent and Accountable Peer Review Training Program
Publicado
DOI
10.5281/zenodo.17833637
Licencia
CC BY 4.0

PREreview of “Incubation of oxycodone craving is associated with CP-AMPAR upregulation in D1 and D2 receptor-expressing medium spiny neurons in nucleus accumbens core and shell”

Summary:

In this article, Mount et al. investigate the effects of sustained oxycodone (oxy) craving on the presence of calcium-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens of male and female rats. This work is very important given the prevalence of the opioid epidemic in the United States and the limited availability of effective treatments and preventive strategies for opioid addiction. Notably, little is known about the synaptic plasticity that occurs during oxycodone craving in the nucleus accumbens (NAc), a central component of the brain’s reward circuitry. Using a combination of behavioral experiments and whole-cell patch-clamp electrophysiology, the authors sought to identify the effects of oxycodone craving on CP-AMPAR expression within subregions of the NAc. They report that oxycodone self-administration and incubation of craving do not differ between male and female rats, and that incubation persists for 30 days following forced abstinence. Furthermore, their ex vivo electrophysiological recordings show that incubation of oxycodone craving is associated with an upregulation of CP-AMPARs in both D1 and D2 medium spiny neurons (MSNs) of the NAc core and shell.

Overall, the work by Mount et al. advances the field’s understanding of NAc synaptic plasticity associated with prolonged oxycodone abstinence, advancing previous studies that have primarily focused on stimulant drugs such as cocaine and methamphetamine. Importantly, the authors expand upon a limited body of research by incorporating data from both male and female rats, as prior studies have typically included only males. While their evidence supports the main claims regarding CP-AMPAR upregulation following incubation of oxycodone craving, additional controls and experiments could further strengthen the study. Likewise, providing additional analyses of existing data could enhance the clarity and impact of the reported findings (see below).

Major Points:

1.     In the drug self-administration section of the methods, the authors describe two different durations for the light cue. Although Supplementary Figure 1J shows no differences in incubation, this result may be influenced by the single female mouse included in the dataset. Conducting an outlier analysis could help strengthen the conclusion that both cue durations yield comparable incubation, particularly in light of the substantial differences in oxycodone infusions observed in Supplementary Figure 1D. Additionally, because the seeking tests for the two groups differed in length (as described in the legend for Supplementary Figure 1), the increased time spent in the chamber by the 4-second group may have introduced differences in extinction that could affect the incubation results. It would also be helpful if the authors clarified their rationale for selecting the 60-minute seeking test rather than the 30-minute version for the D1 and A2a rat experiments.

2.     For Figure 2, it may be helpful if the authors directly compared the incubation scores for AD15 and AD30. This would clarify whether incubation continues to increase during late abstinence or plateaus. Such an analysis seems particularly relevant given that the AD15 seeking test serves as the time point for the electrophysiological recordings, and further analysis would offer a helpful opportunity to substantiate the claim that incubation remains stable from AD15 to AD30 (p. 10). Although a correlation analysis is included in Supplementary Figure 3 to examine plasticity, adding this direct comparison would further strengthen the authors’ conclusions regarding changes in incubation.

Minor Points:

1.     This paper references previous work demonstrating that infusion of a CP-AMPAR antagonist into the NAc core or shell prevents the expression of oxycodone incubation. In the present study, however, the authors report that CP-AMPARs are upregulated in both D1 and A2a MSNs. It would be interesting to determine whether CP-AMPAR signaling in a specific neuronal population (D1, D2, or both) is required for the expression of oxycodone incubation. One potential approach could involve genetically ablating CP-AMPARs in either D1 or A2a rats. Although this question is compelling, I recognize that it might be beyond the scope of the current study.

2.     In the Results section, the incubation score is mentioned but not defined outside of the figure legends. Providing a brief definition in the text when it is first introduced would be helpful for readers.

3.     In Figure 3E, the error bars appear quite large, which may suggest a discrepancy between Figures 3E and 3F. Could the authors clarify whether standard deviation rather than standard error of the mean was used for these plots?

4.     In Figure 3E, the authors use Naspm to confirm the presence of CP-AMPARs in the NAc core. This is a strong control that could further strengthen the study if applied to the additional recordings, including the NAc shell MSNs (Figure 3) as well as the NAc core and shell recordings from D1 and A2a MSNs (Figure 5).

5.     In the Results section, the authors describe performing a paired-pulse ratio analysis (page 10). Including these data—at least in the supplementary figures—would be helpful, as they support the interpretation that the observed effects arise from postsynaptic rather than presynaptic adaptations.

6.     In figure 3H, the traces don’t have a visible scale, could the authors include the corresponding trace in the graph?

7.     The traces shown in Figures 5A and 5B appear visually identical. Could the authors clarify whether these are indeed distinct recordings or if this similarity may have resulted from an inadvertent duplication? Additionally, the saline trace in Figure 5A seems to be missing the sweep corresponding to the 0 mV holding potential.

8.     The preliminary results from the AD1–2 recordings described on page 13 are quite interesting, and incorporating these data—either in the main figures or in the supplementary materials—could further strengthen the manuscript.

Competing interests:

The author declares that they have no competing interests.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.

Comentarios

Escribir un comentario
  1. por Marina Wolf
    Publicado
    Licencia
    CC BY 4.0

    Author response to PREreview (review in regular font, response in italics)

    Summary:

    In this article, Mount et al. investigate the effects of sustained oxycodone (oxy) craving on the presence of calcium-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens of male and female rats. This work is very important given the prevalence of the opioid epidemic in the United States and the limited availability of effective treatments and preventive strategies for opioid addiction. Notably, little is known about the synaptic plasticity that occurs during oxycodone craving in the nucleus accumbens (NAc), a central component of the brain’s reward circuitry. Using a combination of behavioral experiments and whole-cell patch-clamp electrophysiology, the authors sought to identify the effects of oxycodone craving on CP-AMPAR expression within subregions of the NAc. They report that oxycodone self-administration and incubation of craving do not differ between male and female rats, and that incubation persists for 30 days following forced abstinence. Furthermore, their ex vivo electrophysiological recordings show that incubation of oxycodone craving is associated with an upregulation of CP-AMPARs in both D1 and D2 medium spiny neurons (MSNs) of the NAc core and shell.

     Overall, the work by Mount et al. advances the field’s understanding of NAc synaptic plasticity associated with prolonged oxycodone abstinence, advancing previous studies that have primarily focused on stimulant drugs such as cocaine and methamphetamine. Importantly, the authors expand upon a limited body of research by incorporating data from both male and female rats, as prior studies have typically included only males. While their evidence supports the main claims regarding CP-AMPAR upregulation following incubation of oxycodone craving, additional controls and experiments could further strengthen the study. Likewise, providing additional analyses of existing data could enhance the clarity and impact of the reported findings (see below).

    We are grateful for the reviewer’s overall positive assessment of our work.

     Major Points:

    1. In the drug self-administration section of the methods, the authors describe two different durations for the light cue. Although Supplementary Figure 1J shows no differences in incubation, this result may be influenced by the single female mouse included in the dataset. Conducting an outlier analysis could help strengthen the conclusion that both cue durations yield comparable incubation, particularly in light of the substantial differences in oxycodone infusions observed in Supplementary Figure 1D. Additionally, because the seeking tests for the two groups differed in length (as described in the legend for Supplementary Figure 1), the increased time spent in the chamber by the 4-second group may have introduced differences in extinction that could affect the incubation results. It would also be helpful if the authors clarified their rationale for selecting the 60-minute seeking test rather than the 30-minute version for the D1 and A2a rat experiments.

    We appreciate the shared curiosity about how differing cue durations may impact cue learning and incubation. Here we will briefly address the points raised in this comment.  For a more in-depth explanation of our rationale for testing different cue durations, please see Dr. Mount’s dissertation: Engeln, K. A. (2023). Behavioral and synaptic plasticity accompanying incubation of oxycodone craving (Version 4.0) [:unav]. https://doi.org/10.6083/K35695096

    - There was no reason to exclude the female rat in question based on health or drug self-administration history (for clarity we point out that other females were also included in this group).

    - Regarding the difference in seeking test length in the two groups, we took this into account when comparing incubation scores, as was described in the legend to Supp Fig  1J:  “Because seeking test duration differed for these groups (60 min for the 4 s group and 30 min for the 20 s group), analysis was performed on the first 30 min of the test for the 4 s group.”  We did find a typo later in this legend that has been corrected in the updated version of the preprint now in revision for a peer-reviewed journal (5 lines from bottom, there was an incorrect reference to Fig. 3J that has been deleted).  

    - When we began evaluating the effect of shortening the cue duration during self-administration training, we also changed the length of the seeking test to 60 min – the latter change was based on our observation in earlier cohorts that some rats were still poking in the previously active hole towards the end of 30-min seeking tests. We have added this explanation to an update of the preprint now in revision for a peer-reviewed journal. 

    1. For Figure 2, it may be helpful if the authors directly compared the incubation scores for AD15 and AD30. This would clarify whether incubation continues to increase during late abstinence or plateaus. Such an analysis seems particularly relevant given that the AD15 seeking test serves as the time point for the electrophysiological recordings, and further analysis would offer a helpful opportunity to substantiate the claim that incubation remains stable from AD15 to AD30 (p. 10). Although a correlation analysis is included in Supplementary Figure 3 to examine plasticity, adding this direct comparison would further strengthen the authors’ conclusions regarding changes in incubation.

    Graphs showing incubation scores for AD15 and AD30 are presented side by side in Figs 2F and 2G (with the same y axis scale). Although data are split by sex, data points for the two tests are overlapping. Nonetheless, we did run an ANOVA comparing pokes in the previously active hole across AD1, AD15, and AD30 seeking tests. Relative to AD1, active hole responding was significantly elevated on AD15 (p = 0.0164) and AD30 (p = 0.0004), but there was no significant difference in active hole responses betweenAD15 and 30 (p > 0.05). We have added this information to the version of the preprint now in revision for a peer-reviewed journal. In the Fig. 2 legend in the preprint, we focused on male/female comparisons, stating “Male and female rats did not differ in the incubation score on AD15 or AD30 (p>0.05)”. 

    Minor Points:

    1. This paper references previous work demonstrating that infusion of a CP-AMPAR antagonist into the NAc core or shell prevents the expression of oxycodone incubation. In the present study, however, the authors report that CP-AMPARs are upregulated in both D1 and A2a MSNs. It would be interesting to determine whether CP-AMPAR signaling in a specific neuronal population (D1, D2, or both) is required for the expression of oxycodone incubation. One potential approach could involve genetically ablating CP-AMPARs in either D1 or A2a rats. Although this question is compelling, I recognize that it might be beyond the scope of the current study.

    The CP-AMPARs are homomeric GluA1 (Conrad et al, Nature 2008 and other subsequent papers) so this would require genetic ablation of GluA1. However, GluA1 is a critical synaptic protein, so its ablation would likely have substantial effects on synaptic transmission and behavior, even prior to incubation. For example, the AMPARs that dominate in NAc synapses in drug naïve rats are GluA1/GluA2 heteromers.

    1. In the Results section, the incubation score is mentioned but not defined outside of the figure legends. Providing a brief definition in the text when it is first introduced would be helpful for readers.

    Thank you for catching this – we have added the definition in the update of the preprint that is now in revision for a peer-reviewed journal.

    1. In Figure 3E, the error bars appear quite large, which may suggest a discrepancy between Figures 3E and 3F. Could the authors clarify whether standard deviation rather than standard error of the mean was used for these plots?

    The error bars show SEM. We have clarified this in the update of the preprint now in revision for a peer-reviewed journal.

    1. In Figure 3E, the authors use Naspm to confirm the presence of CP-AMPARs in the NAc core. This is a strong control that could further strengthen the study if applied to the additional recordings, including the NAc shell MSNs (Figure 3) as well as the NAc core and shell recordings from D1 and A2a MSNs (Figure 5).

    We appreciate this suggestion.

    1. In the Results section, the authors describe performing a paired-pulse ratio analysis (page 10). Including these data—at least in the supplementary figures—would be helpful, as they support the interpretation that the observed effects arise from postsynaptic rather than presynaptic adaptations.

    These data are provided on p. 10.

    1. In figure 3H, the traces don’t have a visible scale, could the authors include the corresponding trace in the graph?

    Thank you for catching this – we have added the scale bar in the update of the preprint that is now in revision for a peer-reviewed journal.

    1. The traces shown in Figures 5A and 5B appear visually identical. Could the authors clarify whether these are indeed distinct recordings or if this similarity may have resulted from an inadvertent duplication? Additionally, the saline trace in Figure 5A seems to be missing the sweep corresponding to the 0 mV holding potential.

    We are very grateful to the Reviewer for catching these issues – particularly the inadvertent duplication of traces in Figs 5A and 5B! The correct trace has been added to Fig 5B in the update of the preprint that is now in revision for a peer-reviewed journal. Other traces have also been improved.

    1. The preliminary results from the AD1–2 recordings described on page 13 are quite interesting, and incorporating these data—either in the main figures or in the supplementary materials—could further strengthen the manuscript.

    We have added these data in the update of the preprint that is now in revision for a peer-reviewed journal.

    Competing interests

    The author of this comment declares that they have no competing interests.