PREreview of DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates

This review is the result of a virtual, live-streamed preprint journal club organized and hosted by PREreview and the journal Current Research in Neurobiology (CRNEUR) as part of a community-based review pilot (you can read more about the pilot here). The discussion was joined by 12 people in total, including some preprint authors, journal editors and the call facilitators. We thank all participants who contributed to the discussion and made it possible for us to provide feedback to this preprint.

Summary

The manuscript by Mueller and colleagues aims to advance the pathophysiology of pathological anxiety by assessing the effect of increasing activity of the amygdala on anxiety-like behaviors in a nonhuman primate model of anxious temperament. To manipulate the amygdala activity, authors activated excitatory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) receptors with clozapine or deschloroclozapine (DCZ), and used behavioral tests (specifically, the human intruder paradigm) to measure anxiety-like behavior under fear-related contexts. They validate the expression of DREADD receptors through several methods, including PET, histology, and electron microscopy. Additionally, they measured plasma levels of drugs and related metabolites. 

The authors reported an increase in anxiety-like behavior (i.e., increased freezing, etc) when animals expressing activating DREADDs in the amygdala received treatment with clozapine or DCZ. This effect on the behavior remains over a long period (approximately 2 years) post DREADD transfection surgery. Few groups have used activating DREADDs in NHPs, therefore the extensive characterization of DREADD expression, as well as the results of amygdala disinhibition via DREADDs will all be of great interest to a broad community in neuroscience. Also, this manuscript provided new evidence on the functional capacity of DREADD-induced neuronal activation over the long-term. 

Below we list major and minor concerns that were discussed by participants of the journal club, and, where possible, we provide suggestions on how to address those issues.

List of major concerns and feedback:

• The human intruder paradigm (HIP) represents the entirety of the behavior analyzed in this paper. Responding to the HIP has been shown to diminish over repeated exposures, as animals habituate to the task. In this case, the authors show that DREADD-mediated amygdala activation causes an increase in responding to the HIP, so it may be useful to know whether animals’ responding over time was unaltered, or if habituation to the task occurred. If the animals did habituate, this may actually strengthen the argument by showing that DREADD-mediated amygdala activation can ‘rescue’ anxiety-like behavior. An analysis of the animals’ responding after vehicle treatment across the time course of the entire study, and a discussion of this effect, would add to the discussion.

• Overall, the discussion of the DREADD receptor expression observed and its implications for the mechanism underlying the behavioral and physiological results should be expanded or moved into the main text from the supplemental. 

  • Despite targeting the central amygdala, only sparse labeling (~6-9%) of neurons expressed DREADD within this subnucleus. We believe that this point should be addressed clearly in the discussion.

  • The majority of DREADD-expressing cells were instead observed in the basolateral amygdala. The authors speculate that this may be due to the respective cell populations within these regions, and specifically that DREADD uptake and expression may be stronger in glutamatergic neurons. This study is not able to definitely answer whether the behavioral and physiological results observed after DREADD activation are therefore due to the actions of CeA, BLA, a combination of both regions, or downstream regions receiving input from amygdala, but an expansion of the discussion regarding possible mechanisms underlying the observed behavioral results would be beneficial to the reader.

  • It is possible that the retrograde uptake of the virus, although sparse, may have influenced the behavioral results: changes in freezing behavior, for example, may have been influenced by DREADD activation in the motor cortex. This point regarding retrograde uptake of the virus and possible confounding factors should be addressed in the text.

  • We believe that it would be useful to include an analysis that showed the correlation between the degree of receptor expression across amygdala (from histology and/or PET results) and the intensity of changes in anxiety-related behavior exhibited by each subject. 

List of minor concerns and feedback:

• Figure 6 shows that clozapine alone (without DREADD expression) reduces freezing behavior in the HIP, while activation of the amygdala via DREADDs and clozapine increases this behavior. Some discussion of the possible off-target effects of clozapine may serve to strengthen the author’s argument that DREADD-mediated activation of the amygdala is responsible for increasing anxiety-like behavior.

• Some details of the methods should be expanded upon or clarified:

  • The statistics used in this study support the low number of animals used, and within non-human primate neuroscience research these animal numbers and statistical approaches are common and well-supported, but nonetheless the authors should ensure the justification for the low n is clearly explained

  • Similarly, the authors may wish to state that restrictions in animal availability and cost did not allow for a second and possibly stronger control in which a sham surgery would be performed in a separate group of animals

  • When describing the Human Intruder Paradigm, please specify whether the intruder was a individual not known to the animals, and if it was always the same intruder across repetitions of the task

  • Please note whether clozapine (or deschloroclozapine) and vehicle treatments were counterbalanced across animals, and whether the experimenters were blinded to the treatment condition

  • Details about the blood collection used to assess plasma levels of clozapine, deschloroclozapine, and other metabolites should be added to the methods. It would be helpful to specify if the animals were restrained or were previously trained to sit for a blood draw. In the supplemental information, the authors may consider expanding the discussion around the cortisol and ACTH results to include how the stress of blood collection may or may not affect the results

• We include some suggestions to strengthen the figures

  • Representing individual animals as symbols on bar graphs would provide more information on individual variability

  • Please add a key in the figure legends for Supplemental Figures 5, 6, 9, 10, 13, and 15 explaining the abbreviations of the conditions (‘A1’, ‘A2’, etc.)

  • We suggest including a table summarizing all variables analyzed, with individual subjects’ data listed. For example, freezing time in the HIP at baseline and after clozapine or deschloroclozapine treatment, plasma levels of clozapine and deschloroclozapine, level of expression of DREADDs

Concluding remarks

We thank the authors of the preprint for posting their work as such and for agreeing to participate in this pilot. We also thank all participants of the live-preprint journal club for their time and for engaging in the lively discussion that generated this review.

Competing interests

The author declares that they have no competing interests.