Role of anterior insula cortex in context-induced relapse of nicotine-seeking

  1. Hussein Ghareh
  2. Isis Alonso-Lozares
  3. Dustin Schetters
  4. Rae J Herman
  5. Tim S Heistek
  6. Yvar Van Mourik
  7. Philip Jean-Richard-dit-Bressel
  8. Gerald Zernig
  9. Huibert D Mansvelder
  10. Taco J De Vries
  11. Nathan J Marchant

  • Curated by eLife

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    Evaluation Summary:

    This manuscript is of broad interest to readers in the fields of drug addiction and relapse, reinforcement learning and punishment, and those interested in cortical function, particularly the insular cortex. The authors extend a context and punishment-based relapse model to the widely-used drug nicotine and use a number of complementary approaches to support the conclusion that the insular cortex plays a role in nicotine relapse. The experiments were carefully designed and implemented.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Tobacco use is the leading cause of preventable death worldwide, and relapse during abstinence remains the critical barrier to successful treatment of tobacco addiction. During abstinence, environmental contexts associated with nicotine use can induce craving and contribute to relapse. The insular cortex (IC) is thought to be a critical substrate of nicotine addiction and relapse. However, its specific role in context-induced relapse of nicotine-seeking is not fully known. In this study, we report a novel rodent model of context-induced relapse to nicotine-seeking after punishment-imposed abstinence, which models self-imposed abstinence through increasing negative consequences of excessive drug use. Using the neuronal activity marker Fos we find that the anterior (aIC), but not the middle or posterior IC, shows increased activity during context-induced relapse. Combining Fos with retrograde labeling of aIC inputs, we show projections to aIC from contralateral aIC and basolateral amygdala exhibit increased activity during context-induced relapse. Next, we used fiber photometry in aIC and observed phasic increases in aIC activity around nicotine-seeking responses during self-administration, punishment, and the context-induced relapse tests. Next, we used chemogenetic inhibition in both male and female rats to determine whether activity in aIC is necessary for context-induced relapse. We found that chemogenetic inhibition of aIC decreased context-induced nicotine-seeking after either punishment- or extinction-imposed abstinence. These findings highlight the critical role nicotine-associated contexts play in promoting relapse, and they show that aIC activity is critical for this context-induced relapse following both punishment and extinction-imposed abstinence.

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  1. Version published to 10.7554/elife.75609 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    The manuscript by Ghareh et al examines the role of the anterior insula cortex (aIC) in nicotine seeking, punishment-induced abstinence of nicotine seeking and in context induced relapse. The paper used a variety of methods including immediate early gene imaging as a marker of neuronal activity, fibre photometry, inhibition using DREADDs. The paper shows that activity in ipsilateral and contralateral aIC and ipsilateral Bla is elevated in context-induced relapse of punished nicotine-seeking. Population calcium imaging using fibre photometry showed modulated aIC neural activity across nicotine infusion, punishment and relapse test. Although differences in neural activity were not seen during relapse tests across different nose poke options. Silencing the aIC during relapse test reduced relapse after punishment or extinction.

    Strengths:

    Overall the manuscript is of broad interest to the addiction field and researchers interested in insula function. It uses a strong behavioural model to study abstinence and shows clear evidence of relapse following punishment-induced abstinence. It is a model that fits the existing literature on the effects of punishment on drug-seeking.

    The paper uses a variety of methods aimed at providing a thorough picture of aIL neural profile in nicotine-seeking, punishment-induced abstinence of nicotine seeking and context-induced reinstatement of nicotine seeking. There are strong behavioural comparisons for the neural signal including active and inactive nose pokes, nicotine vs nicotine+shock reinforcement, as well as strong neural comparisons including bootstrapping the neural signal, permutation tests and GFP vs hM4Di. The data provide clear evidence using diverse methods for the role of the aIL in context-induced relapse of nicotine-seeking.

    The authors provide important evidence that the aIC regulate relapse of nicotine-seeking similarly whether abstinence was punished- or extinction-induced.

    The discussion is excellent.

    We thank the reviewer for these positive comments.

    Weaknesses:

    Although this is not critical to the paper, having vehicle and GFP controls for clozapine and hm4di would have been preferable. The authors provide a justification for that, which is reasonable, but CNO, which converts to CLZ, does have off-target effects that can only be detected when compared again to the vehicle conditions.

    We do accept this as a valid limitation of our study. We have added additional text in the discussion to temper the conclusions with this limitation in consideration. We have also conducted additional analyses on the inactive nose-pokes and the latency to the first active and inactive nose-pokes on test, further indicating that non-specific behavioural effects are unlikely to explain the findings here.

    It was unclear why the neural signal was modulated during context-induced tests when there were no differences in the neural signal between active nosepokes (followed by nicotine cue or nothing) and inactive nose pokes during the relapse tests. The behaviour clearly shows evidence of relapse. The authors discuss this in terms of targeting different populations of cells. But it is unclear why one would use photometry if the imaging signal could not be used to inform the neural manipulations.

    While we understand this point, we also believe it is important to note that the chemogenetic manipulation data are congruent with the Fos tests. Furthermore, we now more clearly describe the increased calcium activity prior to the active nose-pokes in the relapse tests, to better describe a link between the photometry observations and the chemogenetic manipulations.

    Activity is aligned to nosepoke, but it would be of value to see activity aligned to nicotine alone and nicotine+shock delivery.

    Due to the design of the experiment, all events that we analyzed are necessarily contemporaneous with a nose-poke, which is why in the figure we indicate the nose-poke, and use the difference coloured traces to indicate whether nicotine, nicotine+shock, or nothing occurs afterwards.

    Statistics need to be added to the photometry data. Currently the photometry data are purely descriptive.

    We have added statistical descriptions of the bootstrapping and permutation tests in the results section in the revised manuscript. We have also included the statistical output of these tests in the raw data files.

    The punishment photometry data are quite interesting as the neural signal seems to be similar between the two active nose poke irrespective of whether they lead to nicotine or nothing (active NP>nicotine vs activeNP>nothing). The authors suggest that this is because the nicotine-reinforced active nose poke is modulated, but the data are not so clear. There is a change in the signal (it is no longer biphasic) but the overall increase (assuming identical scale, which I think is reasonable given the scale provided) in the signal seems to change for the active nosepoke that is not reinforced. How punishment affects behaviour on the active nose poke on trials when those nosepoke are not punished is fundamental to understanding the signal and the role of the aIC in this task.

    We appreciate this comment and have added text to the discussion to address this point.

    Reviewer #3 (Public Review):

    Ghareh et al. investigated the role of the anterior insular cortex in context-induced relapse to nicotine seeking after punishment. Notably, the authors extend their previous work on context-induced relapse after punishment to the widely used addictive drug nicotine. The authors use complementary approaches, including Fos immunohistochemistry combined with retrograde tracing, fibre photometry, and chemogenetic inhibition to assess the role of the anterior insular cortex in relapse to nicotine seeking with several different levels of analysis. They show that context-induced relapse to nicotine seeking is associated with increased neuronal activity in the anterior but not middle or posterior insular cortex and with increased activity of ipsilateral anterior insular cortex neurons and contralateral basolateral amygdala neurons that project to the anterior insular cortex. Fiber photometry data show that anterior insular cortex activity increases after nose-pokes that lead to nicotine infusion and punished nose-pokes. Lastly, chemogenetic inhibition of the anterior insular cortex decreases context-induced relapse to nicotine seeking after punishment and extinction.

    Strengths:

    The experiments are well-designed and support the main conclusions of the paper. The authors very nicely show generalization of the context-induced relapse after punishment model to nicotine. On the neurobiological level, it is particularly interesting and informative to juxtapose post-mortem readouts of neuronal activity (Fos immunohistochemistry) with in vivo real-time readouts of neuronal activity (fibre photometry) in awake-behaving rats in the same behavioral procedures. The authors also analyze Fos and CTb expression along the anterior-posterior axis of the anterior insular cortex and basolateral amygdala. An additional strength of the paper is that the authors used chemogenetic inhibition to test the causal role of the anterior insular cortex in context-induced relapse to nicotine seeking after both punishment and extinction.

    Lastly, the authors do an excellent job of pointing out the limitations of their study in the discussion section, which include potential differences in neurobiological substrates depending on route of nicotine administration, exclusion of a vehicle-control group in the chemogenetic experiments, and use of different viral promoters between the fibre photometry and chemogenetic experiments.

    Weaknesses:

    There are two main weaknesses which limit interpretation of the data presented. First, during the punishment phase of the fibre photometry experiment, it is difficult to know which outcome the changes in calcium identified with fibre photometry are due to (e.g., nicotine infusion or footshock). Ideally, appropriate acknowledgement of these limitations in interpretation or inclusion of a yoked control or separate sessions with nicotine infusions or footshock exposure would help address this interpretation issue because this would allow for an analysis that disentangles the complex outcomes.

    We appreciate this comment. Unfortunately, we are unable to disentangle these interpretations due to the experimental design. It would be of interest to determine the extent to which the activity we have observed is dependent on a preceding action (i.e. nose-poke). In the revised manuscript we have added some discussion to address this point.

    Second, with the chemogenetic experiment, the authors observe a decrease in nose pokes in the hM4Di group in Context A (when responding is normally high) but not Context B (when responding is normally low). It is possible that a non-specific effect on responding (e.g. motor or motivational impairment) could be masked in Context B due to a floor effect. Therefore, while the test in Context B is informative, chemogenetic inhibition in another situation where responding is high (e.g. nicotine or food self-administration) would be helpful in the ability to interpret the specificity of hM4Di inhibition of the anterior insular cortex in context-induced relapse to nicotine seeking after punishment or extinction.

    We understand the limitation that is being raised. To address this we have described possible alternative explanations in the limitations section of the discussion. With regard to a potential floor effect, we have conducted further statistical analysis on the inactive nose-pokes, which is relatively high. For example, in punishment punished active nose-pokes are close to zero, and inactive nose-pokes remain stable at around 20 per session. We found no effect of CLZ compared to the rate of inactive responses in the previous sessions. We have updated the manuscript to reflect these points.

  3. eLife

    Evaluation Summary:

    This manuscript is of broad interest to readers in the fields of drug addiction and relapse, reinforcement learning and punishment, and those interested in cortical function, particularly the insular cortex. The authors extend a context and punishment-based relapse model to the widely-used drug nicotine and use a number of complementary approaches to support the conclusion that the insular cortex plays a role in nicotine relapse. The experiments were carefully designed and implemented.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The manuscript by Ghareh et al examines the role of the anterior insula cortex (aIC) in nicotine seeking, punishment-induced abstinence of nicotine seeking and in context induced relapse. The paper used a variety of methods including immediate early gene imaging as a marker of neuronal activity, fibre photometry, inhibition using DREADDs. The paper shows that activity in ipsilateral and contralateral aIC and ipsilateral Bla is elevated in context-induced relapse of punished nicotine-seeking. Population calcium imaging using fibre photometry showed modulated aIC neural activity across nicotine infusion, punishment and relapse test. Although differences in neural activity were not seen during relapse tests across different nose poke options. Silencing the aIC during relapse test reduced relapse after punishment or extinction.

    Strengths:

    Overall the manuscript is of broad interest to the addiction field and researchers interested in insula function. It uses a strong behavioural model to study abstinence and shows clear evidence of relapse following punishment-induced abstinence. It is a model that fits the existing literature on the effects of punishment on drug-seeking.

    The paper uses a variety of methods aimed at providing a thorough picture of aIL neural profile in nicotine-seeking, punishment-induced abstinence of nicotine seeking and context-induced reinstatement of nicotine seeking. There are strong behavioural comparisons for the neural signal including active and inactive nose pokes, nicotine vs nicotine+shock reinforcement, as well as strong neural comparisons including bootstrapping the neural signal, permutation tests and GFP vs hM4Di.

    The data provide clear evidence using diverse methods for the role of the aIL in context-induced relapse of nicotine-seeking.

    The authors provide important evidence that the aIC regulate relapse of nicotine-seeking similarly whether abstinence was punished- or extinction-induced.

    The discussion is excellent.

    Weaknesses:

    Although this is not critical to the paper, having vehicle and GFP controls for clozapine and hm4di would have been preferable. The authors provide a justification for that, which is reasonable, but CNO, which converts to CLZ, does have off-target effects that can only be detected when compared again to the vehicle conditions.

    It was unclear why the neural signal was modulated during context-induced tests when there were no differences in the neural signal between active nosepokes (followed by nicotine cue or nothing) and inactive nose pokes during the relapse tests. The behaviour clearly shows evidence of relapse. The authors discuss this in terms of targeting different populations of cells. But it is unclear why one would use photometry if the imaging signal could not be used to inform the neural manipulations.

    Activity is aligned to nosepoke, but it would be of value to see activity aligned to nicotine alone and nicotine+shock delivery.

    Statistics need to be added to the photometry data. Currently the photometry data are purely descriptive.

    The punishment photometry data are quite interesting as the neural signal seems to be similar between the two active nose poke irrespective of whether they lead to nicotine or nothing (active NP>nicotine vs activeNP>nothing). The authors suggest that this is because the nicotine-reinforced active nose poke is modulated, but the data are not so clear. There is a change in the signal (it is no longer biphasic) but the overall increase (assuming identical scale, which I think is reasonable given the scale provided) in the signal seems to change for the active nosepoke that is not reinforced. How punishment affects behaviour on the active nose poke on trials when those nosepoke are not punished is fundamental to understanding the signal and the role of the aIC in this task.

  5. eLife

    Reviewer #2 (Public Review):

    In their study Ghareh et al. used retrograde tracer with the neuronal activity marker c-fos, fibre-photometry and chemogenetic to functionally characterize the role of the anterior insular cortex (aIC) in a new model of context-induced relapse to nicotine seeking after punishment. They demonstrated an increase of neuronal activity in aIC, and inputs from contralateral aIC and ipsilateral BLA during context-induced relapse to nicotine seeking. Then, they showed an increase of aIC neuronal activity, measured by fibre photometry, during self-administration, punishment, and context-induced relapse. Finally, they demonstrated that chemogenetic inhibition of aIC glutamatergic neurons decreases context-induced nicotine-seeking. The strengths of the manuscript include the longitudinal study of the coding properties of aIC neuronal population during self-administration, punishment and relapse reinforced by the demonstration of the causal role of the aIC in context-induced nicotine relapse. The experiments were carefully designed and implemented. The manuscript is well written and includes interesting and novel findings on the role of aIC neurons in nicotine relapse. However, there are a few methodological and analysis issues. First, a clearer rationale for focusing on contralateral aIC and ipsilateral BLA inputs is needed. Further analyses and clarifications are also needed to fully benefit from the fibre photometry approach.

  6. eLife

    Reviewer #3 (Public Review):

    Ghareh et al. investigated the role of the anterior insular cortex in context-induced relapse to nicotine seeking after punishment. Notably, the authors extend their previous work on context-induced relapse after punishment to the widely used addictive drug nicotine. The authors use complementary approaches, including Fos immunohistochemistry combined with retrograde tracing, fibre photometry, and chemogenetic inhibition to assess the role of the anterior insular cortex in relapse to nicotine seeking with several different levels of analysis. They show that context-induced relapse to nicotine seeking is associated with increased neuronal activity in the anterior but not middle or posterior insular cortex and with increased activity of ipsilateral anterior insular cortex neurons and contralateral basolateral amygdala neurons that project to the anterior insular cortex. Fiber photometry data show that anterior insular cortex activity increases after nose-pokes that lead to nicotine infusion and punished nose-pokes. Lastly, chemogenetic inhibition of the anterior insular cortex decreases context-induced relapse to nicotine seeking after punishment and extinction.

    Strengths:

    The experiments are well-designed and support the main conclusions of the paper. The authors very nicely show generalization of the context-induced relapse after punishment model to nicotine.

    On the neurobiological level, it is particularly interesting and informative to juxtapose post-mortem readouts of neuronal activity (Fos immunohistochemistry) with in vivo real-time readouts of neuronal activity (fibre photometry) in awake-behaving rats in the same behavioral procedures. The authors also analyze Fos and CTb expression along the anterior-posterior axis of the anterior insular cortex and basolateral amygdala. An additional strength of the paper is that the authors used chemogenetic inhibition to test the causal role of the anterior insular cortex in context-induced relapse to nicotine seeking after both punishment and extinction.

    Lastly, the authors do an excellent job of pointing out the limitations of their study in the discussion section, which include potential differences in neurobiological substrates depending on route of nicotine administration, exclusion of a vehicle-control group in the chemogenetic experiments, and use of different viral promoters between the fibre photometry and chemogenetic experiments.

    Weaknesses:

    There are two main weaknesses which limit interpretation of the data presented. First, during the punishment phase of the fibre photometry experiment, it is difficult to know which outcome the changes in calcium identified with fibre photometry are due to (e.g., nicotine infusion or footshock). Ideally, appropriate acknowledgement of these limitations in interpretation or inclusion of a yoked control or separate sessions with nicotine infusions or footshock exposure would help address this interpretation issue because this would allow for an analysis that disentangles the complex outcomes.

    Second, with the chemogenetic experiment, the authors observe a decrease in nose pokes in the hM4Di group in Context A (when responding is normally high) but not Context B (when responding is normally low). It is possible that a non-specific effect on responding (e.g. motor or motivational impairment) could be masked in Context B due to a floor effect. Therefore, while the test in Context B is informative, chemogenetic inhibition in another situation where responding is high (e.g. nicotine or food self-administration) would be helpful in the ability to interpret the specificity of hM4Di inhibition of the anterior insular cortex in context-induced relapse to nicotine seeking after punishment or extinction.

  7. Version published to 10.1101/2021.12.08.471717v1 on bioRxiv