As organisms have evolved, it has been essential that they acquire the means to sense physical and chemical threats in the world around them. One such threat detection system is itch, which accompanies unpleasant sensations that evoke strong urges to scratch and promote learned avoidance behavior (Bautista et al., 2014; Han and Dong, 2014; Ikoma et al., 2006; LaMotte et al., 2014). Orchestrating adaptive behaviors (e.g., scratching an itch, avoidance of active threats) in the future requires rapid routing of information to brain regions that can encode memories and modify behavior based on prior experiences. The central amygdala (CeA) represents a strong candidate for these functions as the CeA is thought to play a critical role in learning and modifying sensory and emotional memories and translating this information into apt adaptive behaviors (Fadok et al., 2018; Gründemann and Lüthi, 2015; LeDoux and Daw, 2018). Recent studies have implicated the CeA in the regulation of itch (Albisetti et al., 2019; Chen et al., 2016; Ehling et al., 2018; Mu et al., 2017), and elevated activity in the CeA has been seen in patients during experimental itch (Papoiu et al., 2014; Vierow et al., 2015). Nevertheless, it is currently unknown how CeA neurons encode and modify the sensory or emotional components of itch. To address these questions, we used optical imaging, activity-dependent labeling, neural tracing and cell activity-specific RNA-sequencing to systematically investigate chloroquine-activated CeA neurons and their projections in eliciting itch and its related comorbidities.

We performed fiber photometry recordings from CeA Vgat neurons in awake, behaving mice to assess the activity of CeA neurons in relation to evoked itch/scratch behaviors. To record real-time Ca²⁺ dynamics in the CeA (Cui et al., 2013), we expressed the genetically encoded calcium indicator, GCaMP6s, in CeA GABAergic neurons using viral delivery of Cre-dependent GCaMP6s in Vgat-IRES-Cre mice (Figure 1a, Figure 1—figure supplement 1a–c). As the majority of CeA neurons are GABAergic (Swanson and Petrovich, 1998), this approach allows us to target the CeA and avoid picking up photometry signals from neighboring BLA neurons, as could occur if we used non Cre-dependent GCaMP6. Subcutaneous injection of chloroquine in the nape of the neck induced scratching behavior and resulted in robust increases in CeA neuronal activity (Figure 1b). This activity commenced with initiation of scratching and stabilized whenever scratching stopped (Figure 1c–e), suggesting that the elevated activity was tightly coupled with the act of scratching. Consistent with these real-time dynamic recordings, activity-dependent mapping studies show robust cFos labeling bilaterally in the CeA following chloroquine injection in the nape of the neck compared to saline-injected mice (Figure 2—figure supplement 1a–c). We observed no significant differences in cFos labeling between right and left CeA (Figure 2—figure supplement 1d).

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These observations provide cellular confirmation of prior reports (Mochizuki et al., 2014; Mochizuki et al., 2003; Papoiu et al., 2013) that indicated a possible role for the CeA in itch processing, but the underlying neural circuitry remains to be identified. If CeA neurons function as a key node in the circuit that tightly regulates sensory and affective component of itch, then their activation should trigger potentiation of the itch-scratching cycle and its aversive state. CeA neurons are molecularly heterogeneous and mediate diverse behaviors generally related to negative affect (John et al., 2015; Kalin et al., 2004; LeDoux, 2003; Ressler and Mayberg, 2007; Roozendaal et al., 2009; Tye et al., 2011), so we reasoned global manipulation of CeA neuronal activity would not provide the specificity needed to test the specific roles of chloroquine-activated neurons. To enable the desired selective manipulation of itch-specific neuronal populations in the CeA, we used ‘Targeted Recombination in Active Populations’ mice (Guenthner et al., 2013). These mice express the tamoxifen-dependent CreER^T2 recombinase from the Fos promoter. CreER^T2 expression is induced in neurons that were recently active. Catalytic activity of CreER^T2 is stabilized in the presence of 4-hydroxytamoxifen (4-OHT), resulting in transgene recombination. By timing the administration of 4-OHT to coincide with recently increased neuronal activity during acute chloroquine stimuli, we can gain permanent genetic access to chloroquine-responsive CeA neurons (aka FosTRAP mice). To test the validity of this approach, we crossed FosTRAP mice to a Cre-dependent tdTomato flox-stop reporter line (Madisen et al., 2010). We injected chloroquine or saline into the nape of the neck, paired with injection of 4-OHT to induce Cre-mediated recombination of the tdTomato in activated (cFos-expressing) neurons (Figure 2a, b). FosTRAPing with chloroquine treatment produced robust tdTomato expression in both the right and left CeA (Figure 2c–e), and small number of neurons in saline-treated controls (Figure 2—figure supplement 1e–g), consistent with our cFos staining results above. This small population of saline TRAPed neurons could be due to the needle stick during the injection itself, and thus could label some pain-responsive CeA neurons. To confirm that the FosTRAPed neurons are specific to the chloroquine-evoked scratching, 1-week post-FosTRAP, we immunostained for c-Fos protein in mice that received an additional chloroquine injection just prior to sacrificing (Figure 2c, f). The majority of the tdTomato-positive CeA FosTRAPed neurons faithfully overlap with cFos-positive cells. These results demonstrate that we can efficiently gain genetic access to neurons that are activated by chloroquine.

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To test whether reactivating chloroquine-responsive CeA neurons can recapitulate itch behaviors, we expressed the Cre-dependent excitatory opsin, ChR2 (AAV5-EF1a-DIO-ChR2-eYFP), or a control virus (AAV5-EF1a-DIO-eYFP) in the right CeA of FosTRAP mice and FosTRAPed with chloroquine treatment as above (Figure 2g). This produces expression of ChR2 specifically in CeA neurons responsive to itch, enabling their selective light-dependent activation. Optogenetic reactivation of FosTRAPed (ChR2⁺) right CeA neurons resulted in significant spontaneous scratching and grooming behaviors compared to pre-stimulation baseline and photostimulation of eYFP-expressing control mice. Interestingly, although ChR2 was FosTRAPed by injecting chloroquine into the nape of the neck, we observed spontaneous scratching and grooming behaviors directed all over the body (data not shown) in a stimulation frequency-dependent manner (Figure 2h, i). Even though some functions of the CeA are lateralized (Carrasquillo and Gereau, 2007), elicitation of scratching behaviors is not lateralized to the right CeA as optical stimulation of FosTRAPed ChR2⁺ neurons in the left CeA also resulted in significant spontaneous scratching behaviors compared to pre-stimulation baseline and photostimulation of eYFP-expressing controls (Figure 2—figure supplement 2). This result is consistent with the observation that chloroquine injection induces cFos expression in left and right CeA (Figure 2—figure supplement 1d). To further confirm these results and as a complementary approach, we expressed the Cre-dependent excitatory DREADD, hM3Dq (AAV5-hSyn-DIO-hM3Dq-mCh), or a control virus (AAV5-hSyn-DIO-mCh) in the CeA of FosTRAP mice. Chemogenetic activation of FosTRAPed CeA neurons also resulted in significant spontaneous scratching behaviors (Figure 2—figure supplement 3e), consistent with the optogenetic results.

In contrast, stimulation of FosTRAPed neurons has no significant effect on hindpaw thermal sensitivity (Figure 2—figure supplement 4b) or licking and biting behaviors. However, stimulation of FosTRAPed neurons slightly increased mechanical sensitivity, suggesting that these neurons can encode generalized scratching behavior and hypersensitivity to mechanical stimuli (Figure 2—figure supplement 4c). These results suggest that FosTRAPed neurons might be involved in nociceptive processing (Neugebauer and Li, 2002). To further determine how reactivation of chloroquine-activated (ChR2⁺) FosTRAPed neurons (hereafter referred to as ‘chloroquine-TRAPed neurons’) can affect ongoing scratching behaviors, we administered chloroquine and optically activated chloroquine-TRAPed CeA neurons. Chloroquine-evoked scratching was potentiated with optical reactivation of CeA chloroquine-TRAPed neurons while no changes were observed in the eYFP controls (Figure 2j). Chemogenetic stimulation of chloroquine-TRAPed neurons produced similar results (Figure 2—figure supplement 3h).

Itch is an aversive sensory experience in humans and rodents (Desbordes et al., 2015; Mochizuki et al., 2015; Mochizuki et al., 2014; Papoiu et al., 2012; Papoiu et al., 2013), and the CeA mediates aversive phenotypes (Carrasquillo and Gereau, 2007; Ciocchi et al., 2010; Ehrlich et al., 2009; Haubensak et al., 2010; Tovote et al., 2016). Therefore, we wanted to assess whether chloroquine-TRAPed CeA neurons encode negative valence associated with itch. We performed closed-loop real-time place-testing (RTPT) to assess affective state, where an animal freely explores two chambers but receives photostimulation of ChR2^+ve chloroquine-TRAPed neurons in only one chamber. Reactivation of chloroquine-TRAPed neurons produced robust place aversion to the stimulated side of the chamber while eYFP-FosTRAPed controls did not (Figure 3b–d), thus demonstrating that chloroquine-activated CeA neurons carry negative reinforcement signals.

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Patients with pruritic skin disorders exhibit heightened anxiety (Ginsburg, 1995), and prior studies have shown CeA as a critical hub in coordinating anxiety states (Ahrens et al., 2018; Shackman and Fox, 2016). Therefore, we evaluated if reactivation of chloroquine-TRAPed CeA neurons can drive anxiety-like behavior using the elevated zero maze (EZM) assay and open-field test (OFT). Optogenetic and chemogenetic reactivation of chloroquine-TRAPed neurons leads to a profound decrease in time spent in the open arms of EZM compared to controls, indicating anxiogenic-like behavioral state (Figure 3e, Figure 3—figure supplement 1b, c). Reactivation of these neurons also leads to decreased time spent in the center during the OFT, further suggesting that these neurons can drive anxiety-like behavior (Figure 3—figure supplement 1d–i). Notably, opto- and chemogenetic reactivation of FosTRAPed neurons did not drive freezing or flight responses in OFT (Figure 3—figure supplement 1e, g), suggesting that these neurons are not involved in fear-like behaviors. We used distance and velocity traveled as surrogate measures of freezing and flight behaviors. Although in our experiments assessing itch and pain behaviors we did not observe obvious freezing or flight behaviors, we did not more formally attempted quantify freezing or flight behaviors. Furthermore, stimulation of these neurons also had no effect on feeding and other appetitive behaviors the CeA is reported to evoke (Douglass et al., 2017; Han et al., 2017; Kim et al., 2017; Li et al., 2017; Figure 3—figure supplements 2 and 3).

Having shown that chloroquine-TRAPed CeA neurons are sufficient to drive itch-related sensory and affective behaviors, we aimed to determine if endogenous activity of these neurons is necessary for itch-related behaviors. We selectively inhibited chloroquine-responsive CeA neurons by expressing the Cre-dependent inhibitory DREADD, hM4Di (AAV5-hSyn-DIO-hM4Di-mCh) or a control virus (AAV5-hSyn-DIO-mCh) in chloroquine-activated CeA neurons of FosTRAP mice (Figure 4a–c). Clozapine N-oxide (CNO) application to ex vivo CeA slices from chloroquine-TRAPed mice decreased neuronal excitability to suprathreshold stimuli (Figure 4d). Chemogenetic inhibition of CeA chloroquine-TRAPed neurons by CNO injection significantly attenuated chloroquine-evoked scratching compared to pre-CNO baseline and compared to mCherry-expressing controls (Figure 4e, f). These results suggest that chloroquine-activated (chloroquine-TRAPed) CeA neurons are necessary for chloroquine-induced scratching behavior. We observed no significant effect on thermal or mechanical sensitivity by inhibiting CeA chloroquine-TRAPed neurons (Figure 2—figure supplement 4). Furthermore, silencing CeA chloroquine-TRAPed neurons did not lead to freezing or flight responses or anxiolytic effects (Figure 3—figure supplement 1j–n). Inhibition of chloroquine-TRAPed neurons also did not affect feeding (Figure 3—figure supplement 2) and appetitive behaviors (Figure 3—figure supplement 3). Because activating CeA chloroquine-TRAPed neurons lead to robust place avoidance, we hypothesized that inhibiting these neurons would block conditioned place aversion (CPA) to chloroquine. We performed CPA to chloroquine in chloroquine-TRAPed mice expressing either hM4Di or mCherry in chloroquine-responsive neurons (Figure 4g). Silencing chloroquine-TRAPed (hM4Di^+ve) CeA neurons with CNO injection in the chloroquine-paired chamber during conditioning blocked CPA to chloroquine, while CNO-treated mCherry controls exhibited CPA to chloroquine, suggesting that these neurons can robustly modulate the aversive component of itch (Figure 4h–k).

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We next sought to understand the circuit context of these chloroquine-activated CeA neurons and explored the downstream nodes that might mediate expression of scratching behaviors. We found that chloroquine-TRAPed CeA neurons send notably dense axonal projections in the ventral periaqueductal gray (vPAG) (Figure 5a–d). We also observed projections to the bed nucleus of stria terminalis (BNST), lateral hypothalamus and faint projection in substantia nigra and parabrachial nucleus (PBN). We confirmed vPAG projections by injection CTB into the vPAG (Figure 5—figure supplement 1a, b) and also by injecting retro Cre DIO GFP in to the vPAG in Vgat Cre mice (Figure 5—figure supplement 1c, d). Injection of RV-GFP into vlPAG of Vgat and Vglut2 Cre mice labeled monosynaptic projections from the CeA consistent with prior work (Avegno et al., 2018; Fadok et al., 2018; Haubensak et al., 2010; Xu et al., 2016; Figure 5—figure supplement 1e–k). Because the vPAG has previously been shown to contribute to pruritic behaviors (Gao et al., 2019; Samineni et al., 2019), we focused our functional studies on this CeA→vPAG circuit. If this CeA→vPAG circuit mediates scratching behaviors elicited by the chloroquine-TRAPed CeA neurons, then stimulating this projection should recapitulate these behaviors. As predicted, photostimulating ChR2-expressing chloroquine-TRAPed CeA neuronal terminals in the vPAG recapitulated spontaneous scratching behaviors (Figure 5e). Activating chloroquine-TRAPed CeA neuronal terminals in the vPAG did not produce freezing or flight responses. To determine if reactivation of chloroquine-TRAPed CeA→vPAG projections (ChR2⁺) can influence ongoing chloroquine-evoked scratching behaviors, we administered chloroquine and optically activated chloroquine-TRAPed CeA→vPAG projections. Chloroquine-evoked scratching was potentiated with optical reactivation of CeA→vPAG projections while no changes were observed in the eYFP controls (Figure 5f). These results show that the CeA→vPAG neuronal circuit is crucial node in mediating pruritic behaviors.

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Lastly, we performed RNA-seq to identify transcriptional profiles of chloroquine-activated CeA cells (Figure 6a). To do this, we TRAPed tdTomato in chloroquine-activated CeA neurons as described above and separated the chloroquine-TRAPed neurons from adjacent tdTomato^-ve cells for comparative RNA-seq analysis (Figure 6—figure supplement 1). Correlation analysis of RNA-seq data revealed chloroquine-TRAPed tdTomato^+ve cells and TRAPed tdTomato^-ve cells are clustered apart from each other (Figure 6—figure supplement 2c). In our sequencing results, we observed that both the tdTomato^+ve and tdTomato^-ve cells expressed Slc32A1 transcript (VGAT, a marker for GABAergic neurons), consistent with the notion that the majority of CeA neurons are GABAergic. Hierarchical clustering analysis of genes shows highly correlated gene expression patterns that show unique expression profiles in FosTRAPed^+ve CeA neurons vs. FosTRAPed^-ve CeA neurons (Figure 6—figure supplement 2d). We identified numerous highly correlated gene clusters based on their expression levels in FosTRAPed^+ve neurons (Figure 6—figure supplement 2e). Subsequent analysis of chloroquine-TRAPed neurons revealed significant enrichment of several unique transcripts in the chloroquine-activated neurons (Figure 6b). Weighted gene correlation network analysis (WGCNA) of genes identified a cluster of upregulated genes 99% correlated and highly significant for chloroquine-activated neurons (Figure 6—figure supplement 2f–h). To link transcriptional profiles of FosTRAPed cells to known CeA functional pathways, we performed pathway analysis. From KEGG and Gene Set Enrichment Analysis (GSEA), we have identified changes in the expression of functionally related candidate genes that are enriched in several pathways (Figure 6c). We have identified significantly enriched CeA candidate genes that might be associated with pruritus regulation, as well as significantly genes expressed at significantly lower levels relative to the non-TRAPed cells that could be involved in the suppression of pruritus. To independently confirm our RNA-seq findings, we performed dual-color fluorescent in situ hybridization (FISH) to visualize mRNA expression of several candidate genes enriched specifically only in the Fos-positive cells induced by pruritic stimuli. We observed considerable overlap between Ntsr2^+ve (75.31% cells per four sections), Gpr88^+ve (69.35% cells per four sections) and Gabrg1^+ve (62.32% cells per four sections) cells with chloroquine activated Fos^+ve cells in the CeA (Figure 6d), which were shown to be significantly enriched in chloroquine-TRAPed tdTomato^+ve cells. To confirm whether this overlap is specific only to the enriched gene cluster, we also assessed the overlap between chloroquine-activated Fos^+ve cells in the CeA and cluster of genes with significantly lower expression in the chloroquine-TRAPed neurons observed from RNA-seq data. We find their partial overlap of Fos^+ve cells in the CeA (Figure 6e) with cells that express Oprm1^+ve (26.47% cells per four sections) Penk^+ve (30.77% cells per four sections) and Chrm1^+ve (36.50% cells per four sections). These results confirm our findings of differentially expressed genes in chloroquine-activated CeA cells and suggesting that further mining of these sequencing data by the community will reveal important new findings related to chloroquine and its comorbidities.

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While there is robust evidence demonstrating the role spinal cord circuits play in driving itch behaviors (Bautista et al., 2014; Han and Dong, 2014; LaMotte et al., 2014; Ross et al., 2010; Sun et al., 2009), the identity and properties of neural circuits in the brain that coordinate pruritic behaviors are still poorly understood. Neural circuits in the CeA are implicated in pruritic process (Chen et al., 2016; Mu et al., 2017; Sanders et al., 2019), but cells and circuits that can alter pruritic processing in the CeA have been unclear. The CeA is well known to regulate a wide variety of aversive (Carrasquillo and Gereau, 2007; Ciocchi et al., 2010; Crock et al., 2012; Ehrlich et al., 2009; Haubensak et al., 2010; Tovote et al., 2016) and appetitive behaviors (Cai et al., 2014; Carter et al., 2013; Douglass et al., 2017; Hardaway et al., 2019; Kim et al., 2017; Robinson et al., 2014; Warlow et al., 2020). Here, we propose a cellular and circuit framework of the CeA in pruritis and its associated affect modulation. By gaining genetic access to neurons that are active specifically during chloroquine-evoked scratching, we were able to selectively identify a diverse repertoire of sensory and aversive behavioral responses mediated by chloroquine-responsive CeA neurons. Activation of chloroquine-responsive neurons in right or left CeA is sufficient to recapitulate spontaneous scratching behaviors, potentiate chloroquine-evoked scratching and produce aversive and anxiety-related behaviors. Furthermore, inhibiting these neurons is sufficient to attenuate ongoing scratching and block its associated aversive component. Our findings reveal the presence of a chloroquine-responsive neuronal population in the CeA, consistent with a recent report (Sanders et al., 2019), that is necessary and sufficient to drive itch-related sensory and affective behaviors. We used both optogenetics and chemogenetics in a complementary manner to confirm these results. Conceptually, our findings seem to reconcile prior work (Chen et al., 2016; Mochizuki et al., 2020; Papoiu et al., 2014; Sanders et al., 2019; Sun et al., 2009; Vierow et al., 2015), suggesting a pivotal role of CeA neurons in pruritic processing. The CeA is known to be involved in threat detection and to produce adaptive responses when organisms encounter threatening conditions (Fadok et al., 2018; Gründemann and Lüthi, 2015; LeDoux and Daw, 2018). These chloroquine-responsive CeA neurons could be a gateway in controlling itch and its associated affective component.

Activation of chloroquine-TRAPed CeA neurons elicits aversive behaviors like place aversion and anxiety-like behaviors, while inhibition of chloroquine-TRAPed CeA neurons produces robust blockade of place aversion to chloroquine. It is well established that the CeA mediates stress-induced anxiety behaviors (Botta et al., 2015; Kalin et al., 2004; Li et al., 2017; Weera et al., 2020), but our results show that silencing chloroquine-TRAPed CeA neurons does not have any effect on basal anxiety-like behavior. Future experiments should address whether these neurons are capable of attenuating anxiety during a variety of threats or stressors. Our findings also imply that chloroquine-activated CeA neurons may also be involved to some extent in regulating nociceptive transmission. Chloroquine-activated CeA neurons that drive pruritic processing could represent a subset or overlapping populations that also process nociceptive information. Our additional experiments also suggest that the CeA chloroquine-responsive neurons do not have any effect on feeding, reward-seeking, motor and freezing behaviors. It is possible that these behaviors are driven by distinct molecularly defined cell types in the CeA (Cai et al., 2014; Carter et al., 2013; Ciocchi et al., 2010; Douglass et al., 2017; Ehrlich et al., 2009; Hardaway et al., 2019; Haubensak et al., 2010; Kim et al., 2017; Robinson et al., 2014; Tovote et al., 2016; Warlow et al., 2020) that are distinct from the chloroquine-activated CeA neurons we have studied here.

We found that chloroquine-activated CeA neurons send functional outputs to the vPAG and activating these CeA→vPAG projections is sufficient to drive scratching. These findings are consistent with recent reports (Gao et al., 2019; Samineni et al., 2019) showing that activating PAG^Vglut2 and PAG^Tac1 neurons produces robust scratching in mice. Based on the results from the present study and reconciling with prior work, a reasonable hypothesis is that information from chloroquine-activated CeA neurons promotes scratching via disinhibition of the PAG^Tac1 output neurons. As activating these PAG populations does not elicit freezing or escape behaviors, it is possible that PAG neurons that process pruritic information may be distinct from the ones that drive freezing, escape or nociceptive behaviors. Based on our findings, pruritic information arriving in PAG neurons originates at least in part via a CeA chloroquine-responsive neuronal population and contributes to the processing and generation of adaptive responses to pruritis. Our RNA-seq data show that CeA neurons are GABAergic, and anatomical tracing data indicate that CeA neurons that project to the PAG are GABAergic inhibitory populations (Figure 5—figure supplement 1). Recent work shows that CeA GABAergic neurons that project to vlPAG can elicit freezing, escape (Haubensak et al., 2010; Tovote et al., 2016), hunting (Han et al., 2017), sleep (Snow et al., 2017) and nociception (Avegno et al., 2018; Li and Sheets, 2018; Yin et al., 2020). In our work, we found that the chloroquine-activated CeA→PAG projections drive pruritic behaviors without evoking freezing or escape behaviors, suggesting that these FosTRAPed CeA→PAG projections are distinctly tuned to elicit scratching. The cellular and molecular identities that distinguish these projections from other CeA neurons are yet to be identified (Steinberg et al., 2020). A critical task in the future will be to identify and characterize how these different modalities of information are differentially processed via these projections and how the postsynaptic neurons in the PAG differentiate this information to transform it into behavioral output. Here, we establish a critical role for inhibitory projections from CeA to PAG in pruritic regulation. Further studies will be required to more fully understand the mechanisms by which this projection drives itch and associated affective behaviors.

Activating CeA FosTRAP neurons resulted in spontaneous scratching and grooming behaviors directed all over the body, and thus were not restricted to the nape of the neck (where the pruritogen injection was administered for the TRAP). Directed behavior related to sensory information could be organized at the level of sensory and motor cortex. Cortical areas have extensive direct descending projections to the dorsal and ventral horn of the spinal cord (Joosten et al., 1992; Liang et al., 2011; Masson et al., 1991; Rouiller et al., 1991). It is not clear how these projections could orchestrate directed scratching behaviors. It is also possible that the CeA is not part of the neural pathways that translate into directed scratching behavior. Neurons in the CeA could receive itch-related information from cortical inputs (Fadok et al., 2018), which could be part of the neural pathways that mediate affective aspects like motivation to scratch an itch or suppress itch to evade any immediate potential threat. Our data show that the CeA sends dense projections to the PAG, which in turn modulates spinal pruritic processing via RVM projections, based on previous reports (Gao et al., 2019; Samineni et al., 2019). There is still much to learn about how this information is organized. Recent work from Gao et al., 2019, suggests that activating Tac1 neurons can drive robust scratching behaviors; this suggests that there could be parallel circuits downstream of the CeA that can evoke and inhibit itch-evoked scratching.

Recent work in the VTA shows that pruritogen-evoked scratching elevates the activity of dopamine neurons, and this elevated activity is required for the hedonic aspects of pruritogen-evoked scratching (Su et al., 2019; Yuan et al., 2018). The VTA is known to send projections to the CeA. It is possible that these projections encode aversive aspects that we have seen in the CeA (Leshan et al., 2010; Zhou et al., 2019). What we cannot parse from our data is whether neurons that encode itch evoked by pruritogens and those that respond to scratching are the same or distinct CeA sub populations. It is possible that there are multiple populations driving sensory and motor aspects of itch-scratch behaviors. We also observed projections from the TRAPed neurons to the BNST, lateral hypothalamus and faint projection in substantia nigra and PBN in addition to the vPAG projections. It is possible that these downstream regions could also play a critical role in different aspects of pruritis. There is now literature suggesting that scratching on the site of pruritogen application can suppress neural activity in spinothalamic neurons (Davidson et al., 2009). In these neurons, activity elicited by pruritogens can be completely abolished by scratching, suggesting that relief of itch by scratching results from suppression of activity in the spinal cord. There are additional studies now showing that supraspinal projections from the PAG and RVM directly modulate this activity in a state-dependent manner (Gao et al., 2019; Samineni et al., 2019). Active inhibition of scratching could take place downstream of CeA when PAG and RVM neurons are engaged in inhibiting ongoing spinal pruritic transmission.

The CeA a is highly molecularly heterogeneous region that is known to express a diverse array of neuropeptides, receptors and cellular machinery that are unique to CeA in integrating and orchestrating neuromodulatory functions (Kim et al., 2017; Zirlinger and Anderson, 2003; Zirlinger et al., 2001). To understand the unique genetic identity of CeA neurons that regulate pruritic behaviors, we performed RNA-seq of chloroquine-activated CeA neurons by TRAPing these neurons with pruritic stimuli. These activity-dependent RNA-Seq data show extensive molecular programs that are selectively enriched in the chloroquine-responsive neurons relative to other cells in CeA. We observe significant enrichment of NTSR2, GPR88 and Gabrg1 in chloroquine-activated neurons, and relatively lower expression of OPRM1, PENK and Chrm1, suggesting that these genes could be critical candidates in regulating pruritis and its associated anxiety. One other interesting observation from our sequencing dataset is the relative enrichment of Prkcd transcript in FosTRAP+ve vs. FosTRAP-ve cells. Prkcd+ve cells in CeA have been shown to be involved in fear and pain processing (Cai et al., 2014; Haubensak et al., 2010; Wilson et al., 2019). It would be interesting to see what role these cells play in pruritic behaviors. It is also possible that the needle stick associated with 4-OHT injection could label a small population of CeA neurons involved in fear or pain processing, and this could impact our sequencing dataset to some extent. In our analysis, we have followed up on six candidate genes, but the dataset we have generated here will be an immensely valuable resource to the neuroscience community interested in the role of CeA neurons in modulation of sensory and affective behaviors.

Sequencing data have been deposited in GEO under accession codes GSE130268.

1. 1. Samineni VK

   (2019) NCBI Gene Expression Omnibus

   ID GSE130268. Transcriptional Idenetity of Itch-activated Central Amygdala Neurons.

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE130268

1. 1. Ahrens S
   2. Wu MV
   3. Furlan A
   4. Hwang G-R
   5. Paik R
   6. Li H
   7. Penzo MA
   8. Tollkuhn J
   9. Li B

   (2018) A Central Extended Amygdala Circuit That Modulates Anxiety

   The Journal of Neuroscience 38:5567–5583.

   https://doi.org/10.1523/JNEUROSCI.0705-18.2018

   • Google Scholar
2. 1. Albisetti GW
   2. Pagani M
   3. Platonova E
   4. Hösli L
   5. Johannssen HC
   6. Fritschy J-M
   7. Wildner H
   8. Zeilhofer HU

   (2019) Dorsal Horn Gastrin-Releasing Peptide Expressing Neurons Transmit Spinal Itch But Not Pain Signals

   The Journal of Neuroscience 39:2238–2250.

   https://doi.org/10.1523/JNEUROSCI.2559-18.2019

   • Google Scholar
3. 1. Arttamangkul S
   2. Torrecilla M
   3. Kobayashi K
   4. Okano H
   5. Williams JT

   (2006) Separation of -Opioid Receptor Desensitization and Internalization: Endogenous Receptors in Primary Neuronal Cultures

   Journal of Neuroscience 26:4118–4125.

   https://doi.org/10.1523/JNEUROSCI.0303-06.2006

   • Google Scholar
4. 1. Avegno EM
   2. Lobell TD
   3. Itoga CA
   4. Baynes BB
   5. Whitaker AM
   6. Weera MM
   7. Edwards S
   8. Middleton JW
   9. Gilpin NW

   (2018) Central Amygdala Circuits Mediate Hyperalgesia in Alcohol-Dependent Rats

   The Journal of Neuroscience 38:7761–7773.

   https://doi.org/10.1523/JNEUROSCI.0483-18.2018

   • Google Scholar
5. 1. Bautista DM
   2. Wilson SR
   3. Hoon MA

   (2014) Why we scratch an itch: the molecules, cells and circuits of itch

   Nature Neuroscience 17:175–182.

   https://doi.org/10.1038/nn.3619

   • Google Scholar
6. 1. Botta P
   2. Demmou L
   3. Kasugai Y
   4. Markovic M
   5. Xu C
   6. Fadok JP
   7. Lu T
   8. Poe MM
   9. Xu L
   10. Cook JM
   11. Rudolph U
   12. Sah P
   13. Ferraguti F
   14. Lüthi A

   (2015) Regulating anxiety with extrasynaptic inhibition

   Nature Neuroscience 18:1493–1500.

   https://doi.org/10.1038/nn.4102

   • Google Scholar
7. 1. Cai H
   2. Haubensak W
   3. Anthony TE
   4. Anderson DJ

   (2014) Central amygdala PKC-δ+ neurons mediate the influence of multiple anorexigenic signals

   Nature Neuroscience 17:1240–1248.

   https://doi.org/10.1038/nn.3767

   • Google Scholar
8. 1. Carrasquillo Y
   2. Gereau RW

   (2007) Activation of the Extracellular Signal-Regulated Kinase in the Amygdala Modulates Pain Perception

   Journal of Neuroscience 27:1543–1551.

   https://doi.org/10.1523/JNEUROSCI.3536-06.2007

   • Google Scholar
9. 1. Carter ME
   2. Soden ME
   3. Zweifel LS
   4. Palmiter RD

   (2013) Genetic identification of a neural circuit that suppresses appetite

   Nature 503:111–114.

   https://doi.org/10.1038/nature12596

   • Google Scholar
10. 1. Chen L
    2. Wang W
    3. Tan T
    4. Han H
    5. Dong Z

    (2016) GABAA Receptors in the Central Nucleus of the Amygdala Are Involved in Pain- and Itch-Related Responses

    The Journal of Pain 17:181–189.

    https://doi.org/10.1016/j.jpain.2015.10.008

    • Google Scholar
11. 1. Ciocchi S
    2. Herry C
    3. Grenier F
    4. Wolff SBE
    5. Letzkus JJ
    6. Vlachos I
    7. Ehrlich I
    8. Sprengel R
    9. Deisseroth K
    10. Stadler MB
    11. Müller C
    12. Lüthi A

    (2010) Encoding of conditioned fear in central amygdala inhibitory circuits

    Nature 468:277–282.

    https://doi.org/10.1038/nature09559

    • Google Scholar
12. 1. Crock LW
    2. Kolber BJ
    3. Morgan CD
    4. Sadler KE
    5. Vogt SK
    6. Bruchas MR
    7. Gereau RW

    (2012) Central Amygdala Metabotropic Glutamate Receptor 5 in the Modulation of Visceral Pain

    Journal of Neuroscience 32:14217–14226.

    https://doi.org/10.1523/JNEUROSCI.1473-12.2012

    • Google Scholar
13. 1. Cui G
    2. Jun SB
    3. Jin X
    4. Pham MD
    5. Vogel SS
    6. Lovinger DM
    7. Costa RM

    (2013) Concurrent activation of striatal direct and indirect pathways during action initiation

    Nature 494:238–242.

    https://doi.org/10.1038/nature11846

    • Google Scholar
14. 1. Davidson S
    2. Zhang X
    3. Khasabov SG
    4. Simone DA
    5. Giesler GJ

    (2009) Relief of itch by scratching: state-dependent inhibition of primate spinothalamic tract neurons

    Nature Neuroscience 12:544–546.

    https://doi.org/10.1038/nn.2292

    • Google Scholar
15. 1. Desbordes G
    2. Li A
    3. Loggia ML
    4. Kim J
    5. Schalock PC
    6. Lerner E
    7. Tran TN
    8. Ring J
    9. Rosen BR
    10. Kaptchuk TJ
    11. Pfab F
    12. Napadow V

    (2015) Evoked itch perception is associated with changes in functional brain connectivity

    NeuroImage: Clinical 7:213–221.

    https://doi.org/10.1016/j.nicl.2014.12.002

    • Google Scholar
16. 1. Douglass AM
    2. Kucukdereli H
    3. Ponserre M
    4. Markovic M
    5. Gründemann J
    6. Strobel C
    7. Alcala Morales PL
    8. Conzelmann K-K
    9. Lüthi A
    10. Klein R

    (2017) Central amygdala circuits modulate food consumption through a positive-valence mechanism

    Nature Neuroscience 20:1384–1394.

    https://doi.org/10.1038/nn.4623

    • Google Scholar
17. 1. Ehling S
    2. Butler A
    3. Thi S
    4. Ghashghaei HT
    5. Bäumer W

    (2018) To scratch an itch: Establishing a mouse model to determine active brain areas involved in acute histaminergic itch

    IBRO Reports 5:67–73.

    https://doi.org/10.1016/j.ibror.2018.10.002

    • Google Scholar
18. 1. Ehrlich I
    2. Humeau Y
    3. Grenier F
    4. Ciocchi S
    5. Herry C
    6. Lüthi A

    (2009) Amygdala Inhibitory Circuits and the Control of Fear Memory

    Neuron 62:757–771.

    https://doi.org/10.1016/j.neuron.2009.05.026

    • Google Scholar
19. 1. Fadok JP
    2. Markovic M
    3. Tovote P
    4. Lüthi A

    (2018) New perspectives on central amygdala function

    Current Opinion in Neurobiology 49:141–147.

    https://doi.org/10.1016/j.conb.2018.02.009

    • Google Scholar
20. 1. Gao Z-R
    2. Chen W-Z
    3. Liu M-Z
    4. Chen X-J
    5. Wan L
    6. Zhang X-Y
    7. Yuan L
    8. Lin J-K
    9. Wang M
    10. Zhou L
    11. Xu X-H
    12. Sun Y-G

    (2019) Tac1-Expressing Neurons in the Periaqueductal Gray Facilitate the Itch-Scratching Cycle via Descending Regulation

    Neuron 101:45–59.

    https://doi.org/10.1016/j.neuron.2018.11.010

    • Google Scholar
21. 1. Ginsburg IH

    (1995) Psychological and Psychophysiological Aspects of Psoriasis

    Dermatologic Clinics 13:793–804.

    https://doi.org/10.1016/S0733-8635(18)30043-3

    • Google Scholar
22. 1. Gründemann J
    2. Lüthi A

    (2015) Ensemble coding in amygdala circuits for associative learning

    Current Opinion in Neurobiology 35:200–206.

    https://doi.org/10.1016/j.conb.2015.10.005

    • Google Scholar
23. 1. Guenthner CJ
    2. Miyamichi K
    3. Yang HH
    4. Heller HC
    5. Luo L

    (2013) Permanent Genetic Access to Transiently Active Neurons via TRAP: Targeted Recombination in Active Populations

    Neuron 78:773–784.

    https://doi.org/10.1016/j.neuron.2013.03.025

    • Google Scholar
24. 1. Guez-Barber D
    2. Fanous S
    3. Harvey BK
    4. Zhang Y
    5. Lehrmann E
    6. Becker KG
    7. Picciotto MR
    8. Hope BT

    (2012) FACS purification of immunolabeled cell types from adult rat brain

    Journal of Neuroscience Methods 203:10–18.

    https://doi.org/10.1016/j.jneumeth.2011.08.045

    • Google Scholar
25. 1. Han W
    2. Tellez LA
    3. Rangel MJ
    4. Motta SC
    5. Zhang X
    6. Perez IO
    7. Canteras NS
    8. Shammah-Lagnado SJ
    9. van den Pol AN
    10. de Araujo IE

    (2017) Integrated Control of Predatory Hunting by the Central Nucleus of the Amygdala

    Cell 168:e318–e324.

    https://doi.org/10.1016/j.cell.2016.12.027

    • Google Scholar
26. 1. Han L
    2. Dong X

    (2014) Itch Mechanisms and Circuits

    Annual Review of Biophysics 43:331–355.

    https://doi.org/10.1146/annurev-biophys-051013-022826

    • Google Scholar
27. 1. Hardaway JA
    2. Halladay LR
    3. Mazzone CM
    4. Pati D
    5. Bloodgood DW
    6. Kim M
    7. Jensen J
    8. DiBerto JF
    9. Boyt KM
    10. Shiddapur A
    11. Erfani A
    12. Hon OJ
    13. Neira S
    14. Stanhope CM
    15. Sugam JA
    16. Saddoris MP
    17. Tipton G
    18. McElligott Z
    19. Jhou TC
    20. Stuber GD
    21. Bruchas MR
    22. Bulik CM
    23. Holmes A
    24. Kash TL

    (2019) Central Amygdala Prepronociceptin-Expressing Neurons Mediate Palatable Food Consumption and Reward

    Neuron 102:e1037–e1052.

    https://doi.org/10.1016/j.neuron.2019.03.037

    • Google Scholar
28. 1. Haubensak W
    2. Kunwar PS
    3. Cai H
    4. Ciocchi S
    5. Wall NR
    6. Ponnusamy R
    7. Biag J
    8. Dong H-W
    9. Deisseroth K
    10. Callaway EM
    11. Fanselow MS
    12. Lüthi A
    13. Anderson DJ

    (2010) Genetic dissection of an amygdala microcircuit that gates conditioned fear

    Nature 468:270–276.

    https://doi.org/10.1038/nature09553

    • Google Scholar
29. 1. Hempel CM
    2. Sugino K
    3. Nelson SB

    (2007) A manual method for the purification of fluorescently labeled neurons from the mammalian brain

    Nature Protocols 2:2924–2929.

    https://doi.org/10.1038/nprot.2007.416

    • Google Scholar
30. 1. Ikoma A
    2. Steinhoff M
    3. Ständer S
    4. Yosipovitch G
    5. Schmelz M

    (2006) The neurobiology of itch

    Nature Reviews Neuroscience 7:535–547.

    https://doi.org/10.1038/nrn1950

    • Google Scholar
31. 1. John CS
    2. Sypek EI
    3. Carlezon WA
    4. Cohen BM
    5. Öngür D
    6. Bechtholt AJ

    (2015) Blockade of the GLT-1 Transporter in the Central Nucleus of the Amygdala Induces both Anxiety and Depressive-Like Symptoms

    Neuropsychopharmacology 40:1700–1708.

    https://doi.org/10.1038/npp.2015.16

    • Google Scholar
32. 1. Joosten EAJ
    2. Schuitman RL
    3. Vermelis MEJ
    4. Dederen PJWC

    (1992) Postnatal development of the ipsilateral corticospinal component in rat spinal cord: A light and electron microscopic anterograde HRP study

    The Journal of Comparative Neurology 326:133–146.

    https://doi.org/10.1002/cne.903260112

    • Google Scholar
33. 1. Kalin NH
    2. Shelton SE
    3. Davidson RJ

    (2004) The Role of the Central Nucleus of the Amygdala in Mediating Fear and Anxiety in the Primate

    Journal of Neuroscience 24:5506–5515.

    https://doi.org/10.1523/JNEUROSCI.0292-04.2004

    • Google Scholar
34. 1. Kim J
    2. Zhang X
    3. Muralidhar S
    4. LeBlanc SA
    5. Tonegawa S

    (2017) Basolateral to Central Amygdala Neural Circuits for Appetitive Behaviors

    Neuron 93:e1465–e1479.

    https://doi.org/10.1016/j.neuron.2017.02.034

    • Google Scholar
35. 1. LaMotte RH
    2. Dong X
    3. Ringkamp M

    (2014) Sensory neurons and circuits mediating itch

    Nature Reviews Neuroscience 15:19–31.

    https://doi.org/10.1038/nrn3641

    • Google Scholar
36. 1. Land BB
    2. Bruchas MR
    3. Schattauer S
    4. Giardino WJ
    5. Aita M
    6. Messinger D
    7. Hnasko TS
    8. Palmiter RD
    9. Chavkin C

    (2009) Activation of the kappa opioid receptor in the dorsal raphe nucleus mediates the aversive effects of stress and reinstates drug seeking

    PNAS 106:19168–19173.

    https://doi.org/10.1073/pnas.0910705106

    • Google Scholar
37. 1. LeDoux J

    (2003) The emotional brain, fear, and the amygdala

    Cellular and Molecular Neurobiology 23:727–738.

    https://doi.org/10.1023/A:1025048802629

    • Google Scholar
38. 1. LeDoux J
    2. Daw ND

    (2018) Surviving threats: neural circuit and computational implications of a new taxonomy of defensive behaviour

    Nature Reviews Neuroscience 19:269–282.

    https://doi.org/10.1038/nrn.2018.22

    • Google Scholar
39. 1. Leshan RL
    2. Opland DM
    3. Louis GW
    4. Leinninger GM
    5. Patterson CM
    6. Rhodes CJ
    7. Munzberg H
    8. Myers MG

    (2010) Ventral Tegmental Area Leptin Receptor Neurons Specifically Project to and Regulate Cocaine- and Amphetamine-Regulated Transcript Neurons of the Extended Central Amygdala

    Journal of Neuroscience 30:5713–5723.

    https://doi.org/10.1523/JNEUROSCI.1001-10.2010

    • Google Scholar
40. 1. Li M-J
    2. Liu L-Y
    3. Chen L
    4. Cai J
    5. Wan Y
    6. Xing G-G

    (2017) Chronic stress exacerbates neuropathic pain via the integration of stress-affect–related information with nociceptive information in the central nucleus of the amygdala

    Pain 158:717–739.

    https://doi.org/10.1097/j.pain.0000000000000827

    • Google Scholar
41. 1. Li JN
    2. Sheets PL

    (2018) The central amygdala to periaqueductal gray pathway comprises intrinsically distinct neurons differentially affected in a model of inflammatory pain

    The Journal of Physiology 596:6289–6305.

    https://doi.org/10.1113/JP276935

    • PubMed
    • Google Scholar
42. 1. Liang H
    2. Paxinos G
    3. Watson C

    (2011) Projections from the brain to the spinal cord in the mouse

    Brain Structure and Function 215:159–186.

    https://doi.org/10.1007/s00429-010-0281-x

    • Google Scholar
43. 1. Madisen L
    2. Zwingman TA
    3. Sunkin SM
    4. Oh SW
    5. Zariwala HA
    6. Gu H
    7. Ng LL
    8. Palmiter RD
    9. Hawrylycz MJ
    10. Jones AR
    11. Lein ES
    12. Zeng H

    (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain

    Nature Neuroscience 13:133–140.

    https://doi.org/10.1038/nn.2467

    • Google Scholar
44. 1. Masson RL
    2. Sparkes ML
    3. Ritz LA

    (1991) Descending projections to the rat sacrocaudal spinal cord

    The Journal of Comparative Neurology 307:120–130.

    https://doi.org/10.1002/cne.903070111

    • Google Scholar
45. 1. Mochizuki H
    2. Tashiro M
    3. Kano M
    4. Sakurada Y
    5. Itoh M
    6. Yanai K

    (2003) Imaging of central itch modulation in the human brain using positron emission tomography

    Pain 105:339–346.

    https://doi.org/10.1016/S0304-3959(03)00249-5

    • Google Scholar
46. Book

    1. Mochizuki H
    2. Papoiu ADP
    3. Yosipovitch G

    (2014)

    Brain Processing of Itch and Scratching

    In: Carstens E, Akiyama T, editors. Itch: Mechanisms and Treatment. CRC Press/Taylor & Francis. pp. 1806–1811.

    • Google Scholar
47. 1. Mochizuki H
    2. Papoiu ADP
    3. Nattkemper LA
    4. Lin AC
    5. Kraft RA
    6. Coghill RC
    7. Yosipovitch G

    (2015) Scratching Induces Overactivity in Motor-Related Regions and Reward System in Chronic Itch Patients

    Journal of Investigative Dermatology 135:2814–2823.

    https://doi.org/10.1038/jid.2015.223

    • Google Scholar
48. 1. Mochizuki H
    2. Hernandez L
    3. Yosipovitch G
    4. Sadato N
    5. Kakigi R

    (2020) The Amygdala Network for Processing Itch in Human Brains

    Acta Dermato Venereologica 100:adv00345.

    https://doi.org/10.2340/00015555-3703

    • Google Scholar
49. 1. Montana MC
    2. Conrardy BA
    3. Cavallone LF
    4. Kolber BJ
    5. Rao LK
    6. Greco SC
    7. Gereau RW

    (2011) Metabotropic glutamate receptor 5 antagonism with fenobam: examination of analgesic tolerance and side effect profile in mice

    Anesthesiology 115:1239–1250.

    https://doi.org/10.1097/ALN.0b013e318238c051

    • PubMed
    • Google Scholar
50. 1. Mu D
    2. Deng J
    3. Liu K-F
    4. Wu Z-Y
    5. Shi Y-F
    6. Guo W-M
    7. Mao Q-Q
    8. Liu X-J
    9. Li H
    10. Sun Y-G

    (2017) A central neural circuit for itch sensation

    Science 357:695–699.

    https://doi.org/10.1126/science.aaf4918

    • Google Scholar
51. 1. Neugebauer V
    2. Li W

    (2002) Processing of Nociceptive Mechanical and Thermal Information in Central Amygdala Neurons With Knee-Joint Input

    Journal of Neurophysiology 87:103–112.

    https://doi.org/10.1152/jn.00264.2001

    • Google Scholar
52. 1. O'Brien DE
    2. Brenner DS
    3. Gutmann DH
    4. Gereau RW

    (2013) Assessment of Pain and Itch Behavior in a Mouse Model of Neurofibromatosis Type 1

    The Journal of Pain 14:628–637.

    https://doi.org/10.1016/j.jpain.2013.01.770

    • Google Scholar
53. 1. Papoiu ADP
    2. Coghill RC
    3. Kraft RA
    4. Wang H
    5. Yosipovitch G

    (2012) A tale of two itches Common features and notable differences in brain activation evoked by cowhage and histamine induced itch

    NeuroImage 59:3611–3623.

    https://doi.org/10.1016/j.neuroimage.2011.10.099

    • Google Scholar
54. 1. Papoiu ADP
    2. Nattkemper LA
    3. Sanders KM
    4. Kraft RA
    5. Chan Y-H
    6. Coghill RC
    7. Yosipovitch G

    (2013) Brain’s Reward Circuits Mediate Itch Relief A Functional MRI Study of Active Scratching

    PLOS ONE 8:e82389.

    https://doi.org/10.1371/journal.pone.0082389

    • Google Scholar
55. 1. Papoiu ADP
    2. Emerson NM
    3. Patel TS
    4. Kraft RA
    5. Valdes-Rodriguez R
    6. Nattkemper LA
    7. Coghill RC
    8. Yosipovitch G

    (2014) Voxel-based morphometry and arterial spin labeling fMRI reveal neuropathic and neuroplastic features of brain processing of itch in end-stage renal disease

    Journal of Neurophysiology 112:1729–1738.

    https://doi.org/10.1152/jn.00827.2013

    • Google Scholar
56. 1. Ressler KJ
    2. Mayberg HS

    (2007) Targeting abnormal neural circuits in mood and anxiety disorders: from the laboratory to the clinic

    Nature Neuroscience 10:1116–1124.

    https://doi.org/10.1038/nn1944

    • Google Scholar
57. 1. Robinson MJF
    2. Warlow SM
    3. Berridge KC

    (2014) Optogenetic Excitation of Central Amygdala Amplifies and Narrows Incentive Motivation to Pursue One Reward Above Another

    Journal of Neuroscience 34:16567–16580.

    https://doi.org/10.1523/JNEUROSCI.2013-14.2014

    • Google Scholar
58. 1. Roozendaal B
    2. McEwen BS
    3. Chattarji S

    (2009) Stress, memory and the amygdala

    Nature Reviews Neuroscience 10:423–433.

    https://doi.org/10.1038/nrn2651

    • Google Scholar
59. 1. Ross SE
    2. Mardinly AR
    3. McCord AE
    4. Zurawski J
    5. Cohen S
    6. Jung C
    7. Hu L
    8. Mok SI
    9. Shah A
    10. Savner EM
    11. Tolias C
    12. Corfas R
    13. Chen S
    14. Inquimbert P
    15. Xu Y
    16. McInnes RR
    17. Rice FL
    18. Corfas G
    19. Ma Q
    20. Woolf CJ
    21. Greenberg ME

    (2010) Loss of Inhibitory Interneurons in the Dorsal Spinal Cord and Elevated Itch in Bhlhb5 Mutant Mice

    Neuron 65:886–898.

    https://doi.org/10.1016/j.neuron.2010.02.025

    • Google Scholar
60. 1. Rouiller EM
    2. Liang F
    3. Moret V
    4. Wiesendanger M

    (1991) Trajectory of redirected corticospinal axons after unilateral lesion of the sensorimotor cortex in neonatal rat; A phaseolus vulgaris-leucoagglutinin (PHA-L) tracing study

    Experimental Neurology 114:53–65.

    https://doi.org/10.1016/0014-4886(91)90084-P

    • Google Scholar
61. 1. Samineni VK
    2. Grajales-Reyes JG
    3. Copits BA
    4. O'Brien DE
    5. Trigg SL
    6. Gomez AM
    7. Bruchas MR
    8. Gereau RW

    (2017a) Divergent modulation of nociception by glutamatergic and GABAergic neuronal subpopulations in the periaqueductal gray

    Eneuro 4:ENEURO.0129-16.2017.

    https://doi.org/10.1523/ENEURO.0129-16.2017

    • PubMed
    • Google Scholar
62. 1. Samineni VK
    2. Mickle AD
    3. Yoon J
    4. Grajales-Reyes JG
    5. Pullen MY
    6. Crawford KE
    7. Noh KN
    8. Gereau GB
    9. Vogt SK
    10. Lai HH
    11. Rogers JA
    12. Gereau RW

    (2017b) Optogenetic silencing of nociceptive primary afferents reduces evoked and ongoing bladder pain

    Scientific Reports 7:15865.

    https://doi.org/10.1038/s41598-017-16129-3

    • PubMed
    • Google Scholar
63. 1. Samineni VK
    2. Grajales-Reyes JG
    3. Sundaram SS
    4. Yoo JJ
    5. Gereau RW

    (2019) Cell type-specific modulation of sensory and affective components of itch in the periaqueductal gray

    Nature Communications 10:4356.

    https://doi.org/10.1038/s41467-019-12316-0

    • Google Scholar
64. 1. Sanders KM
    2. Sakai K
    3. Henry TD
    4. Hashimoto T
    5. Akiyama T

    (2019) A Subpopulation of Amygdala Neurons Mediates the Affective Component of Itch

    The Journal of Neuroscience 39:3345–3356.

    https://doi.org/10.1523/JNEUROSCI.2759-18.2019

    • Google Scholar
65. 1. Seo D-oh
    2. Funderburk SC
    3. Bhatti DL
    4. Motard LE
    5. Newbold D
    6. Girven KS
    7. McCall JG
    8. Krashes M
    9. Sparta DR
    10. Bruchas MR

    (2016) A GABAergic Projection from the Centromedial Nuclei of the Amygdala to Ventromedial Prefrontal Cortex Modulates Reward Behavior

    The Journal of Neuroscience 36:10831–10842.

    https://doi.org/10.1523/JNEUROSCI.1164-16.2016

    • Google Scholar
66. 1. Shackman AJ
    2. Fox AS

    (2016) Contributions of the Central Extended Amygdala to Fear and Anxiety

    Journal of Neuroscience 36:8050–8063.

    https://doi.org/10.1523/JNEUROSCI.0982-16.2016

    • Google Scholar
67. 1. Shin G
    2. Gomez AM
    3. Al-Hasani R
    4. Jeong YR
    5. Kim J
    6. Xie Z
    7. Banks A
    8. Lee SM
    9. Han SY
    10. Yoo CJ
    11. Lee J-L
    12. Lee SH
    13. Kurniawan J
    14. Tureb J
    15. Guo Z
    16. Yoon J
    17. Park S-I
    18. Bang SY
    19. Nam Y
    20. Walicki MC
    21. Samineni VK
    22. Mickle AD
    23. Lee K
    24. Heo SY
    25. McCall JG
    26. Pan T
    27. Wang L
    28. Feng X
    29. Kim T-il
    30. Kim JK
    31. Li Y
    32. Huang Y
    33. Gereau RW
    34. Ha JS
    35. Bruchas MR
    36. Rogers JA

    (2017) Flexible Near-Field Wireless Optoelectronics as Subdermal Implants for Broad Applications in Optogenetics

    Neuron 93:509–521.

    https://doi.org/10.1016/j.neuron.2016.12.031

    • Google Scholar
68. 1. Snow MB
    2. Fraigne JJ
    3. Thibault-Messier G
    4. Chuen VL
    5. Thomasian A
    6. Horner RL
    7. Peever J

    (2017) GABA Cells in the Central Nucleus of the Amygdala Promote Cataplexy

    The Journal of Neuroscience 37:4007–4022.

    https://doi.org/10.1523/JNEUROSCI.4070-15.2017

    • Google Scholar
69. 1. Steinberg EE
    2. Gore F
    3. Heifets BD
    4. Taylor MD
    5. Norville ZC
    6. Beier KT
    7. Földy C
    8. Lerner TN
    9. Luo L
    10. Deisseroth K
    11. Malenka RC

    (2020) Amygdala-Midbrain Connections Modulate Appetitive and Aversive Learning

    Neuron 106:e1029–e1043.

    https://doi.org/10.1016/j.neuron.2020.03.016

    • Google Scholar
70. 1. Su X-Y
    2. Chen M
    3. Yuan Y
    4. Li Y
    5. Guo S-S
    6. Luo H-Q
    7. Huang C
    8. Sun W
    9. Li Y
    10. Zhu MX
    11. Liu M-G
    12. Hu J
    13. Xu T-L

    (2019) Central Processing of Itch in the Midbrain Reward Center

    Neuron 102:858–872.

    https://doi.org/10.1016/j.neuron.2019.03.030

    • Google Scholar
71. 1. Sun Y-G
    2. Zhao Z-Q
    3. Meng X-L
    4. Yin J
    5. Liu X-Y
    6. Chen Z-F

    (2009) Cellular Basis of Itch Sensation

    Science 325:1531–1534.

    https://doi.org/10.1126/science.1174868

    • Google Scholar
72. 1. Swanson LW
    2. Petrovich GD

    (1998) What is the amygdala?

    Trends in Neurosciences 21:323–331.

    https://doi.org/10.1016/S0166-2236(98)01265-X

    • Google Scholar
73. 1. Tovote P
    2. Esposito MS
    3. Botta P
    4. Chaudun F
    5. Fadok JP
    6. Markovic M
    7. Wolff SBE
    8. Ramakrishnan C
    9. Fenno L
    10. Deisseroth K
    11. Herry C
    12. Arber S
    13. Lüthi A

    (2016) Midbrain circuits for defensive behaviour

    Nature 534:206–212.

    https://doi.org/10.1038/nature17996

    • Google Scholar
74. 1. Tye KM
    2. Prakash R
    3. Kim S-Y
    4. Fenno LE
    5. Grosenick L
    6. Zarabi H
    7. Thompson KR
    8. Gradinaru V
    9. Ramakrishnan C
    10. Deisseroth K

    (2011) Amygdala circuitry mediating reversible and bidirectional control of anxiety

    Nature 471:358–362.

    https://doi.org/10.1038/nature09820

    • Google Scholar
75. 1. Tzschentke TM

    (2007) Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade

    Addiction Biology 12:227–462.

    https://doi.org/10.1111/j.1369-1600.2007.00070.x

    • Google Scholar
76. 1. Valtcheva MV
    2. Samineni VK
    3. Golden JP
    4. Gereau RW
    5. Davidson S

    (2015) Enhanced Nonpeptidergic Intraepidermal Fiber Density and an Expanded Subset of Chloroquine-Responsive Trigeminal Neurons in a Mouse Model of Dry Skin Itch

    The Journal of Pain 16:346–356.

    https://doi.org/10.1016/j.jpain.2015.01.005

    • Google Scholar
77. 1. Vierow V
    2. Forster C
    3. Vogelgsang R
    4. Dörfler A
    5. Handwerker H

    (2015) Cerebral Networks Linked to Itch-related Sensations Induced by Histamine and Capsaicin

    Acta Dermato Venereologica 95:645–652.

    https://doi.org/10.2340/00015555-2006

    • Google Scholar
78. 1. Warlow SM
    2. Naffziger EE
    3. Berridge KC

    (2020) The central amygdala recruits mesocorticolimbic circuitry for pursuit of reward or pain

    Nature Communications 11:2716.

    https://doi.org/10.1038/s41467-020-16407-1

    • Google Scholar
79. 1. Weera MM
    2. Schreiber AL
    3. Avegno EM
    4. Gilpin NW

    (2020) The role of central amygdala corticotropin-releasing factor in predator odor stress-induced avoidance behavior and escalated alcohol drinking in rats

    Neuropharmacology 166:107979.

    https://doi.org/10.1016/j.neuropharm.2020.107979

    • Google Scholar
80. 1. Wilson TD
    2. Valdivia S
    3. Khan A
    4. Ahn H-S
    5. Adke AP
    6. Martinez Gonzalez S
    7. Sugimura YK
    8. Carrasquillo Y

    (2019) Dual and Opposing Functions of the Central Amygdala in the Modulation of Pain

    Cell Reports 29:e335–e346.

    https://doi.org/10.1016/j.celrep.2019.09.011

    • Google Scholar
81. 1. Xu C
    2. Krabbe S
    3. Gründemann J
    4. Botta P
    5. Fadok JP
    6. Osakada F
    7. Saur D
    8. Grewe BF
    9. Schnitzer MJ
    10. Callaway EM
    11. Lüthi A

    (2016) Distinct Hippocampal Pathways Mediate Dissociable Roles of Context in Memory Retrieval

    Cell 167:e916–e972.

    https://doi.org/10.1016/j.cell.2016.09.051

    • Google Scholar
82. 1. Yin W
    2. Mei L
    3. Sun T
    4. Wang Y
    5. Li J
    6. Chen C
    7. Farzinpour Z
    8. Mao Y
    9. Tao W
    10. Li J
    11. Xie W
    12. Zhang Z

    (2020) A Central Amygdala–Ventrolateral Periaqueductal Gray Matter Pathway for Pain in a Mouse Model of Depression-like Behavior

    Anesthesiology 132:1175–1196.

    https://doi.org/10.1097/ALN.0000000000003133

    • Google Scholar
83. 1. Yuan L
    2. Liang T-Y
    3. Deng J
    4. Sun Y-G

    (2018) Dynamics and Functional Role of Dopaminergic Neurons in the Ventral Tegmental Area during Itch Processing

    The Journal of Neuroscience 38:9856–9869.

    https://doi.org/10.1523/JNEUROSCI.1483-18.2018

    • Google Scholar
84. 1. Zhou Z
    2. Liu X
    3. Chen S
    4. Zhang Z
    5. Liu Y
    6. Montardy Q
    7. Tang Y
    8. Wei P
    9. Liu N
    10. Li L
    11. Song R
    12. Lai J
    13. He X
    14. Chen C
    15. Bi G
    16. Feng G
    17. Xu F
    18. Wang L

    (2019) A VTA GABAergic Neural Circuit Mediates Visually Evoked Innate Defensive Responses

    Neuron 103:e476–e488.

    https://doi.org/10.1016/j.neuron.2019.05.027

    • Google Scholar
85. 1. Zirlinger M
    2. Kreiman G
    3. Anderson DJ

    (2001) Amygdala-enriched genes identified by microarray technology are restricted to specific amygdaloid subnuclei

    PNAS 98:5270–5275.

    https://doi.org/10.1073/pnas.091094698

    • Google Scholar
86. 1. Zirlinger M
    2. Anderson D

    (2003) Molecular dissection of the amygdala and its relevance to autism

    Genes, Brain and Behavior 2:282–294.

    https://doi.org/10.1034/j.1601-183X.2003.00039.x

    • Google Scholar

Author details

1. Vijay K Samineni

   Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9491-2793

2. Jose G Grajales-Reyes

   1. Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States
   2. Medical Scientist Training Program, Washington University School of Medicine, St. Louis, United States
   3. Neuroscience Program, Washington University School of Medicine, St. Louis, United States

   Contribution

   Funding acquisition, Validation, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

3. Gary E Grajales-Reyes

   Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. Eric Tycksen

   Genome Technology Access Center, Washington University School of Medicine, Seattle, United States

   Contribution

   Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6362-0141

5. Bryan A Copits

   Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3732-890X

6. Christian Pedersen

   Department of Biomedical Engineering, University of Washington, Seattle, United States

   Contribution

   Software, Formal analysis

   Competing interests

   No competing interests declared

7. Edem S Ankudey

   Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Julian N Sackey

   Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Sienna B Sewell

   Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

10. Michael R Bruchas

    1. Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States
    2. Departments of Anesthesiology and Pharmacology, University of Washington, Seattle, United States
    3. Departmentsof Neuroscience and Biomedical Engineering, Washington University School of Medicine, St.Louis, United States

    Contribution

    Resources, Software, Supervision

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4713-7816

11. Robert W Gereau

    1. Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States
    2. Department of Biomedical Engineering, University of Washington, Seattle, United States
    3. Departmentsof Neuroscience and Biomedical Engineering, Washington University School of Medicine, St.Louis, United States

    Contribution

    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Project administration, Writing - review and editing

    For correspondence

    gereaur@wustl.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-5428-4251

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