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.

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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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