1. Author Response:

    **Reviewer #1: **

    Samineni et al. seek to identify and characterize the brain mechanisms responsible for itch-related behaviors. Previous work by this group and others showed that mouse CeA contains itch-responsive neurons. Here the authors set out to determine the molecular and circuit identity of these neurons, their necessity and sufficiency in controlling scratching behavior and itch-related affective components. Using photometry in Vgat-IRES-Cre animals, they show that GABAergic neurons in CeA are active during scratching behavior. In subsequent experiments, scratch-responsive neurons are TRAPed (with scratching behavior elicited by pruritogenic chloroquine injections) and later manipulated using optogenetics and DREADD to test their necessity and sufficiency in scratching behavior and other known CeA-dependent behaviors. Scratching bouts are optogenetically driven with or without chloroquine, suggesting that the neurons are sufficient to elicit this behavior. Optogenetic stimulation is also used in a closed-loop real time assay and zero plus maze to show that chloroquine-TRAPed CeA neurons encode aversive affect and anxiety-related behaviors. Inhibitory DREADD is used to show that TRAPed neurons are required for choroquine-mediated itch behaviors and aversive affect elicited by chloroquine. Appetitive studies show that manipulation of chloroquine-TRAPed neurons does not affect free feeding or food seeking. Viral tracing studies show a connection between the CeA and vPAG and optogenetic manipulations of axon terminals in this circuit reproduces findings with TRAPed CeA neuronal manipulations. Finally, TRAPed neurons are isolated and sequenced in an effort to identify their unique molecular profiles. These results strongly suggest that a subtype(s) of CeA neurons are activated by chloroquine and are important for both scratching behavior and affective aspects of the behavior, while not being involved in appetitive behaviors. However, the use of terms like 'active avoidance' is misleading based on the assays used and interpretation of some of the findings is muddied somewhat by missing or inadequately described control data.

    We thank the reviewer for the thoughtful comments on our work. We do understand how our use of “active avoidance” can lead to confusion. Itch is an aversive sensory experience. In mice, pruritic stimuli (chloroquine and histamine) can produce robust place aversion (Mu and Sun, 2017 and Samineni et al, 2019). We interpreted this learned avoidance to pruritic stimuli as 'active avoidance'. As you pointed out, this can lead to confusion in interpreting our results. To mitigate any confusion, we have now removed any reference to active avoidance in the manuscript. We have also addressed other minor issues raised as requested.

    Reviewer #2:

    The neurological pathways that give rise to the distinct response to irritation of the skin are largely unknown. This study investigates the neurons in a region of the brain well known to be, in part, responsible for assignment of positive and negative valence to sensory information, the amygdala. The data in this study clearly establish an important role of the central area of the amygdala in initiating itch. It provides several lines of evidence for this conclusion using different molecular genetic approaches. The weaknesses of the study are minor.

    We have addressed the minor issues identified by the reviewer, including clarifying why we chose Vgat neurons for our fiber photometry experiments and the nature of additional projection fields from the itch-activated neurons.

    Reviewer #3:

    Samineni et al. provide a beautiful insight into the mouse circuitries of itching in the Central Amygdala, a region of the brain that has apart from its role in pain, received ample attention for its role in feeding and freezing/escape to threat behavior. The manuscript provides an impressive amount experimental evidence, combining activity dependent gene expression with expression of genetically encoded calcium indicators, fluorescent proteins, optogenetic and chemogenetic tools, fiberoptometry and behavioral readouts. With these they identify a subpopulation of GABAergic neurons in the central amygdala that are activated by neck-applied chloroquine-induced itch (as witnessed by the presence of specific scratching in the neck). They show how their specific optogenetic reactivation (in the absence of chloroquine) induces 1). (non-directed all over the body) scratching 2). Real-time place aversion, and reduced spending in open arm of elevated zero maze. And they show how specific chemogenetic inhibition in the presence of chloroquine reduces scratching and real-time place aversion . They then go further to show by fluorescence axonal projections of these neurons in the vPAG and how optogenetic activation of these projections in the vPAG also induces (non-directed) scratching behavior. Finally they identify the genetic blueprint of these neurons with FACS. The experiments all well performed and provide convincing evidence for the implication of neurons in the CeA in sensitivity to itch and activity of scratching. It stands out for a rich combination of diverse state of the art technical approaches that are appropriate applied to answer the questions at hand.

    In its completeness, the manuscript raises an important number of open questions in the field, and I would like to encourage the authors to identify these more clearly in their discussion, as they could set out a pathway along which this field may develop further.

    We appreciate the overall positive assessment, and the suggestion to expand the discussion of a number of issues. We have made significant changes to the discussion to address the findings regarding spontaneous, non-directed scratching induced by reactivation of the chloroquine-trapped neurons, the relationship of genes found to be enriched in itch-activated CeA neurons and those identified as important markers of cells involved in other behaviors (such as pain, fear), and the various projection fields observed in itch-activated neurons with a discussion of their relationship to various aspects of the itch/scratch cycle.

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  2. Reviewer #3 (Public Review):

    Samineni et al. provide a beautiful insight into the mouse circuitries of itching in the Central Amygdala, a region of the brain that has apart from its role in pain, received ample attention for its role in feeding and freezing/escape to threat behavior. The manuscript provides an impressive amount experimental evidence, combining activity dependent gene expression with expression of genetically encoded calcium indicators, fluorescent proteins, optogenetic and chemogenetic tools, fiberoptometry and behavioral readouts. With these they identify a subpopulation of GABAergic neurons in the central amygdala that are activated by neck-applied chloroquine-induced itch (as witnessed by the presence of specific scratching in the neck). They show how their specific optogenetic reactivation (in the absence of chloroquine) induces 1). (non-directed all over the body) scratching 2). Real-time place aversion, and reduced spending in open arm of elevated zero maze. And they show how specific chemogenetic inhibition in the presence of chloroquine reduces scratching and real-time place aversion . They then go further to show by fluorescence axonal projections of these neurons in the vPAG and how optogenetic activation of these projections in the vPAG also induces (non-directed) scratching behavior. Finally they identify the genetic blueprint of these neurons with FACS. The experiments all well performed and provide convincing evidence for the implication of neurons in the CeA in sensitivity to itch and activity of scratching. It stands out for a rich combination of diverse state of the art technical approaches that are appropriate applied to answer the questions at hand.

    In its completeness, the manuscript raises an important number of open questions in the field, and I would like to encourage the authors to identify these more clearly in their discussion, as they could set out a pathway along which this field may develop further.

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  3. Reviewer #2 (Public Review):

    The neurological pathways that give rise to the distinct response to irritation of the skin are largely unknown. This study investigates the neurons in a region of the brain well known to be, in part, responsible for assignment of positive and negative valence to sensory information, the amygdala. The data in this study clearly establish an important role of the central area of the amygdala in initiating itch. It provides several lines of evidence for this conclusion using different molecular genetic approaches. The weaknesses of the study are minor.

    Read the original source
    Was this evaluation helpful?
  4. Reviewer #1 (Public Review):

    Samineni et al. seek to identify and characterize the brain mechanisms responsible for itch-related behaviors. Previous work by this group and others showed that mouse CeA contains itch-responsive neurons. Here the authors set out to determine the molecular and circuit identity of these neurons, their necessity and sufficiency in controlling scratching behavior and itch-related affective components. Using photometry in Vgat-IRES-Cre animals, they show that GABAergic neurons in CeA are active during scratching behavior. In subsequent experiments, scratch-responsive neurons are TRAPed (with scratching behavior elicited by pruritogenic chloroquine injections) and later manipulated using optogenetics and DREADD to test their necessity and sufficiency in scratching behavior and other known CeA-dependent behaviors. Scratching bouts are optogenetically driven with or without chloroquine, suggesting that the neurons are sufficient to elicit this behavior. Optogenetic stimulation is also used in a closed-loop real time assay and zero plus maze to show that chloroquine-TRAPed CeA neurons encode aversive affect and anxiety-related behaviors. Inhibitory DREADD is used to show that TRAPed neurons are required for choroquine-mediated itch behaviors and aversive affect elicited by chloroquine. Appetitive studies show that manipulation of chloroquine-TRAPed neurons does not affect free feeding or food seeking. Viral tracing studies show a connection between the CeA and vPAG and optogenetic manipulations of axon terminals in this circuit reproduces findings with TRAPed CeA neuronal manipulations. Finally, TRAPed neurons are isolated and sequenced in an effort to identify their unique molecular profiles. These results strongly suggest that a subtype(s) of CeA neurons are activated by chloroquine and are important for both scratching behavior and affective aspects of the behavior, while not being involved in appetitive behaviors. However, the use of terms like 'active avoidance' is misleading based on the assays used and interpretation of some of the findings is muddied somewhat by missing or inadequately described control data.

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  5. Evaluation Summary:

    This work will be of general interest to neuroscientists, especially those studying how the brain processes itch stimuli and controls itch-related behavior. The authors show that specific cells in the central amygdala (and their communication with other parts of the brain) play an important role in itching (pruritic) behavior. Overall, the authors provides several lines of evidence to support their conclusions.

    (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. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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