Active avoidance responses (ARs) are instrumental behaviors that prevent harm. Adaptive ARs may contribute to active coping, whereas maladaptive avoidance habits are implicated in anxiety and obsessive-compulsive disorders. The AR learning mechanism has remained elusive, as successful avoidance trials produce no obvious reinforcer. We used a novel outcome-devaluation procedure in rats to show that ARs are positively reinforced by response-produced feedback cues that develop into safety signals during training. Males were sensitive to feedback devaluation after moderate training, but not overtraining, consistent with a transition from goal-directed to habitual avoidance. Using chemogenetics and feedback devaluation, we also show that goal-directed vs. habitual ARs depend on dorsomedial vs. dorsolateral striatum, suggesting a significant overlap between the mechanisms of avoidance and rewarded instrumental behavior. Females were insensitive to feedbackdevaluation due to a remarkable context-dependence of counterconditioning. However, degrading the contingency between avoidance and feedback suggests that both sexes rely on safety signals to perform goal-directed ARs.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Itch is an unpleasant sensation that elicits robust scratching and aversive experience. However, the identity of the cells and neural circuits that organize this information remains elusive. Here, we show the necessity and sufficiency of chloroquine-activated neurons in the central amygdala (CeA) for both itch sensation and associated aversion. Further, we show that chloroquine-activated CeA neurons play important roles in itch-related comorbidities, including anxiety-like behaviors, but not in some aversive and appetitive behaviors previously ascribed to CeA neurons. RNA-sequencing of chloroquine-activated CeA neurons identified several differentially expressed genes as well as potential key signaling pathways in regulating pruritis. Finally, viral tracing experiments demonstrate that these neurons send projections to the ventral periaqueductal gray that are critical in modulation of itch. These findings reveal a cellular and circuit signature of CeA neurons orchestrating behavioral and affective responses to pruritus in mice. Introduction 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. Results 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). Figure 1 with 1 supplement see all Download asset Open asset Neural dynamics of itch activated with central amygdala (CeA) neurons. (a) Scheme demonstrating viral injection strategy and fiber placement to record CeA Vgat neural activity in response to chloroquine. (b) Raw Ca2+ dynamics recorded from CeA Vgat neurons and their relationship to chloroquine-evoked scratching bouts (orange bars). (c) Heatmap showing Ca2+ dynamics of all trials of Vgat+ve vlPAG neurons relative to the initiation of chloroquine-evoked scratching bouts (time zero). (d) Averaged GCaMP6s fluorescence signal of CeA Vgat neurons showing rapid increases in fluorescence on the initiation of scratching bouts. Trace plotted as mean (blue line) ± SEM (gray shading), and the vertical line indicates initiation of scratching bouts. (e) Chloroquine-evoked scratching resulted in a significant increase in CeA Vgat neuronal activity as measured by this change in GCaMP6s fluorescence (N = 8, t test, t = 5.923, df = 14, p<0.0001). 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 CreERT2 recombinase from the Fos promoter. CreERT2 expression is induced in neurons that were recently active. Catalytic activity of CreERT2 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. Figure 2 with 4 supplements see all Download asset Open asset Chloroquine-activated central amygdala (CeA) neurons can drive pruritic behaviors. (a) FosTRAP strategy to selectively label chloroquine-activated neurons in the CeA. (b) Scheme illustrating experimental strategy. (c) FosTRAPing with chloroquine-evoked scratching produces robust tdTomato expression in the CeA. Colocalization of chloroquine-TRAPed neurons (red) in the CeA with cFos immunoreactivity (green) following a second administration of chloroquine 7 days post. Scale bar = 85 and 250 μm. (d) Quantification of the number of FosTRAPed neurons in left and right CeA after chloroquine injection. n = 3 per group. t test, t = 0.4339, df = 2, p=0.70. (e) Rostro-caudal distribution of chloroquine-TRAPed CeA neurons after chloroquine injection. (f) Colocalization of chloroquine-activated cFos with tdTomato+ve chloroquine-TRAPed neurons. Relative percentages of Fos+ve neurons that are tdTomato+ve and tdTomato+ve neurons that are Fos+ve. n = 6 per group. t test, t = 2.04 df = 10, p=0.048. (g) Scheme to selectively express optogenetic constructs in chloroquine-TRAPed CeA neurons. Illustration and representative section showing fiber optic placement above FosTRAPed CeA neurons expressing ChR2-eYFP (green). Scale bar, 100 μm. (h) Photostimulation (20 Hz) of chloroquine-TRAPed CeA neurons produces robust spontaneous scratching. n = 6–11 per group. Pre vs. Stim, F (1,30) = 3; eYFP vs. ChR2, F (1,14) = 3.24, p=0.0001, ANOVA and Bonferroni's for post hoc tests. (i) Increases in scratching are frequency dependent. n = 6 per group. (j) Optical activation of chloroquine-TRAPed CeA neurons potentiates chloroquine-evoked scratching while no changes were observed in control mice. n = 7 per group. Pre vs. Stim, F (1,12) = 33.15; BL vs. Stim in ChR2, F (1, 6) = 6.915, p=0.0391, ANOVA and Bonferroni's for post hoc tests. 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. Figure 3 with 3 supplements see all Download asset Open asset Chloroquine-activated central amygdala (CeA) neurons are negatively reinforcing. (a) Illustration of strategy to express ChR2/eYFP selectively in chloroquine-TRAPed neurons of the CeA. Experimental schematic of closed loop real-time assay and elevated zero maze (EZM). (b) Real-time place aversion assay with spatial location heatmaps of ChR2 and eYFP mice during closed loop optical stimulation. (c) Total time spent and (d) distance traveled in the photostimulation-paired chamber for ChR2 and eYFP mice. n = 7 per group, t test, t = 2.806, df = 12, p=0.0159, t test, t = 0.7510, df = 12, p=0.4142. (e) Representative occupancy heatmap showing spatial location in the EZM of a control mouse (eYFP) and a mouse injected with DIO-ChR2. (f) Optogenetic activation of chloroquine-TRAPed CeA neurons causes a significant reduction in time spent in open arms in EZM. Light stimulation was delivered entire time mice were on EZM n = 6–10 per group. t test, t = 5.922, df = 12, p=0.0086. 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). Figure 4 Download asset Open asset Inhibiting chloroquine-activated central amygdala (CeA) neurons impairs aversive learning associated with itch. (a) Illustration of strategy to express inhibitory DREADDs selectively in chloroquine-TRAPed neurons of the CeA. (b) Experimental timeline to FosTRAP DREADDs in CeA neurons. (c) Representative section showing chloroquine-TRAPed CeA neurons expressing hM4Di-mCherry (red). Scale bar, 75 μm. (d) Infrared DIC image of CeA chloroquine-TRAPed neurons expressing hM4Di-mCherry. In hM4Di+ve CeA neurons, clozapine N-oxide (CNO) bath application decreased neuronal excitability to suprathreshold stimuli. (e) Chemogenetic inhibition of chloroquine-TRAPed CeA neurons leads to a significant reduction in chloroquine-evoked scratching. CNO has no effect on chloroquine-evoked scratching in control mice expressing mCherry. n = 8–9 per group. p=0.0011, ANOVA and Bonferroni's for post hoc tests. (f) Heatmap showing averaged chloroquine-evoked scratching bouts pre- and post-CNO in mice expressing hM4Di in CeA. (g) Schematic and timeline of conditioned place aversion experimental design with chemogenetic silencing. Representative heatmap showing spatial location of a control mouse injected with the DIO-mCh (h) and the DIO-hM4Di DREADD virus (i), pre- and post-chloroquine conditioning. (j) Change in chamber occupancy time in the chloroquine-paired chamber compared to the saline-paired chamber after chemogenetic silencing. n = 11 per group. p=0.044, ANOVA and Bonferroni's for post hoc tests. (k) Distance traveled in chloroquine-paired chamber did not differ pre- and post-conditioning in mCh and hM4Di mice. n = 0.769 per group, ANOVA and Bonferroni's for post hoc tests. 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. Figure 5 with 1 supplement see all Download asset Open asset Identification of the downstream circuit of chloroquine-activated central amygdala (CeA) neurons. (a) Scheme showing expression of ChR2 in chloroquine-TRAPed CeA neurons and their axonal photostimulation in the ventral periaqueductal gray (vPAG). (b) FosTRAPed CeA neurons expressing ChR2-eYFP. Scale bar, 125 μm. (c, d) Chloroquine-TRAPed ChR2+ve CeA axonal terminals ramify densely in the vPAG. Scale bar, 100 and 25 μm. (e) Optogenetic stimulation of FosTRAPed ChR2+ve axonal projections from CeA in the vPAG resulted in significant spontaneous scratching, whereas photostimulation had no effect on scratching in control mice. Pre vs. Stim, F (1,12) = 33.15, p<0.0001, n = 5–7 per group, ANOVA and Bonferroni's for post hoc tests. (f) Optical activation of chloroquine-TRAPed CeA neurons potentiates chloroquine-evoked scratching while no changes were observed in control mice. n = 7 per group. BL vs. Stim in eYFP, F (1,6) = 0.019, p=0.8924; BL vs. Stim in ChR2, F (1, 6) = 9.109, p=0.0235, ANOVA and Bonferroni's for post hoc tests. 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. Figure 6 with 2 supplements see all Download asset Open asset Cell-type-specific transcriptomic profiling of chloroquine-activated central amygdala (CeA) cells. (a) Experimental workflow outlining fluorescence-activated cell sorting (FACS) of the FosTRAPed tdTomato+ve and tdTomato-ve CeA neurons for whole-cell transcriptomics analyses. (b) Volcano plot of log2-fold change (x axis) and p values (y axis) showing the transcripts that are differentially expressed in the chloroquine-TRAPed tdTomato+ve CeA cells. Significantly differentially expressed genes are color coded, and genes that have p≤0.001 are indicated on the plot. (c) Candidate genes identified by fold change in expression of genes in significantly enriched KEGG pathways from the FosTRAPed tdTomato+ve CeA cells. (d) Multiplexed fluorescent in situ hybridization (FISH) was used in validating the expression of NTSR2, GPR88 and GABARG1 in itch-activated Fos+ve CeA cells. We observed considerable overlap between NTSR2+ve (75.31% cells), GPR88+ve (69.35%) and Gabrg1+ve (62.32% cells) cells with itch-activated Fos+ve cells in the CeA. (e) Multiplexed FISH was used to verify the overlap of OPRM1, Penk and Chrm1 in itch-activated Fos+ve CeA cells. We find their partial overlap of Fos+ve cells in the CeA with cells that express OPRM1+ve (26.47% cells), Penk+ve (30.77% cells) and Chrm1+ve (36.50% cells). Right corner of each image shows magnification of the inset (yellow box). Discussion 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

In animal models that mimic human third-trimester fetal development, ethanol causes substantial cellular apoptosis in the brain, but for most brain structures, the extent of permanent neuron loss that persists into adulthood is unknown. We injected ethanol into C57BL/6J mouse pups at postnatal day 7 (P7) to model human late-gestation ethanol toxicity, and then used stereological methods to investigate adult cell numbers in several subcortical neurotransmitter systems that project extensively in the forebrain to regulate arousal states. Ethanol treatment caused especially large reductions (34–42%) in the cholinergic cells of the basal forebrain, including cholinergic cells in the medial septal/vertical diagonal band nuclei (Ch1/Ch2) and in the horizontal diagonal band/substantia innominata/nucleus basalis nuclei (Ch3/Ch4). Cell loss was also present in non-cholinergic basal forebrain cells, as demonstrated by 34% reduction of parvalbumin-immunolabeled GABA cells and 25% reduction of total Nissl-stained neurons in the Ch1/Ch2 region. In contrast, cholinergic cells in the striatum were reduced only 12% by ethanol, and those of the brainstem pedunculopontine/lateral dorsal tegmental nuclei (Ch5/Ch6) were not significantly reduced. Similarly, ethanol did not significantly reduce dopamine cells of the ventral tegmental area/substantia nigra or serotonin cells in the dorsal raphe nucleus. Orexin (hypocretin) cells in the hypothalamus showed a modest reduction (14%). Our findings indicate that the basal forebrain is especially vulnerable to alcohol exposure in the late gestational period. Reduction of cholinergic and GABAergic projection neurons from the basal forebrain that regulate forebrain arousal may contribute to the behavioral and cognitive deficits associated with neonatal ethanol exposure.

In signaled active avoidance (SigAA), rats learn to suppress Pavlovian freezing and emit actions to remove threats and prevent footshocks. SigAA is critical for understanding aversively motivated instrumental behavior and anxiety-related active coping. However, with standard protocols ∼25% of rats exhibit high freezing and poor avoidance. This has dampened enthusiasm for the paradigm and stalled progress. We demonstrate that reducing shock imminence with long-duration warning signals leads to greater freezing suppression and perfect avoidance in all subjects. This suggests that instrumental SigAA mechanisms evolved to cope with distant harm and protocols that promote inflexible Pavlovian reactions are poorly designed to study avoidance.

Local translation can support memory consolidation by supplying new proteins to synapses undergoing plasticity. Translation in adult forebrain dendrites is an established mechanism of synaptic plasticity and is regulated by learning, yet there is no evidence for learning-regulated protein synthesis in adult forebrain axons, which have traditionally been believed to be incapable of translation. Here, we show that axons in the adult rat amygdala contain translation machinery, and use translating ribosome affinity purification (TRAP) with RNASeq to identify mRNAs in cortical axons projecting to the amygdala, over 1200 of which were regulated during consolidation of associative memory. Mitochondrial and translation-related genes were upregulated, whereas synaptic, cytoskeletal, and myelin-related genes were downregulated; the opposite effects were observed in the cortex. Our results demonstrate that axonal translation occurs in the adult forebrain and is altered after learning, supporting the likelihood that local translation is more a rule than an exception in neuronal processes.

Norepinephrine (NE) plays a central role in the acquisition of aversive learning via actions in the lateral nucleus of the amygdala (LA) [1, 2]. However, the function of NE in expression of aversively-conditioned responses has not been established. Given the role of the central nucleus of the amygdala (CeA) in the expression of such behaviors [3-5], and the presence of NE axons projections in this brain nucleus [6], we assessed the effects of NE activity in the CeA on behavioral expression using receptor-specific pharmacology and cell- and projection-specific chemogenetic manipulations. We found that inhibition and activation of locus coeruleus (LC) neurons decreases and increases freezing to aversively conditioned cues, respectively. We then show that locally inhibiting or activating LC terminals in CeA is sufficient to achieve this bidirectional modulation of defensive reactions. These findings support the hypothesis that LC projections to CeA are critical for the expression of defensive responses elicited by conditioned threats.

Local translation can support memory consolidation by supplying new proteins to synapses undergoing plasticity. Translation in adult forebrain dendrites is an established mechanism of synaptic plasticity and is regulated by learning, yet there is no evidence for learning-regulated protein synthesis in adult forebrain axons, which have traditionally been believed to be incapable of translation. Here we show that axons in the adult rat amygdala contain translation machinery, and use translating ribosome affinity purification (TRAP) with RNASeq to identify mRNAs in cortical axons projecting to the amygdala, over 1200 of which were regulated during consolidation of associative memory. Mitochondrial and translation-related genes were upregulated, whereas synaptic, cytoskeletal, and myelin-related genes were downregulated; the opposite effects were observed in the cortex. Our results demonstrate that learning-regulated axonal translation occurs in the adult forebrain, and support the likelihood that local translation is more a rule than an exception in neuronal processes.

Signaled active avoidance (SigAA) is the key experimental procedure for studying the acquisition of instrumental responses toward conditioned threat cues. Traditional analytic approaches (e.g., general linear model) often obfuscate important individual differences, although individual differences in learned responses characterize both animal and human learning data. However, individual differences models (e.g., latent growth curve modeling) typically require large samples and onerous computational methods. Here, we present an analytic methodology that enables the detection of individual differences in SigAA performance at a high accuracy, even when a single animal is included in the data set (i.e., n =1 level). We further show an online software that enables the easy application of our method to any SigAA data set.

Through the process of neuronal differentiation, newly born neurons change from simple, spherical cells to complex, sprawling cells with many highly branched processes. One of the first stages in this process is neurite initiation, wherein cytoskeletal modifications facilitate membrane protrusion and extension from the cell body. Hundreds of actin modulators and microtubule-binding proteins are known to be involved in this process, but relatively little is known about how upstream regulators bring these complex networks together at discrete locations to produce neurites. Here, we show that Myristoylated alanine-rich C kinase substrate (MARCKS) participates in this process. Marcks-/- cortical neurons extend fewer neurites and have less complex neurite arborization patterns. We use an in vitro proteomics screen to identify MARCKS interactors in developing neurites and characterize an interaction between MARCKS and a CDC42-centered network. While the presence of MARCKS does not affect whole brain levels of activated or total CDC42, we propose that MARCKS is uniquely positioned to regulate CDC42 localization and interactions within specialized cellular compartments, such as nascent neurites.

The creation of auditory threat Pavlovian memory requires an initial learning stage in which a neutral conditioned stimulus (CS), such as a tone, is paired with an aversive one (US), such as a shock. In this phase, the CS acquires the capacity of predicting the occurrence of the US and therefore elicits conditioned defense responses. Norepinephrine (NE), through β-adrenergic receptors in the amygdala, enhances threat memory by facilitating the acquisition of the CS-US association, but the nature of this effect has not been described. Here we show that NE release, induced by the footshock of the first conditioning trial, promotes the subsequent enhancement of learning. Consequently, blocking NE transmission disrupts multitrial but not one-trial conditioning. We further found that increasing the time between the conditioning trials eliminates the amplificatory effect of NE. Similarly, an unsignaled footshock delivered in a separate context immediately before conditioning can enhance learning. These results help define the conditions under which NE should and should not be expected to alter threat processing and fill an important gap in the understanding of the neural processes relevant to the pathophysiology of stress and anxiety disorders.

The neural mechanisms through which a Pavlovian conditioned stimulus (CS) elicits innate defense responses are well understood. But a Pavlovian CS can also invigorate ongoing instrumental responding, as shown by studies of aversive Pavlovian-to-instrumental transfer (PIT). While the neural circuitry of appetitive PIT has been studied extensively, little is known about the brain mechanisms of aversive PIT. We recently showed the central amygdala (CeA) is essential for aversive PIT. In the current studies, using pharmacology and designer receptors in rodents, we demonstrate that noradrenergic (NE) activity negatively regulates PIT via brainstem locus coeruleus (LC) activity and LC projections to CeA. Our results provide evidence for a novel pathway through which response modulation occurs between brainstem neuromodulatory systems and CeA to invigorate adaptive behavior in the face of threat.

Much of the early research in aversive learning concerned motivation and reinforcement in avoidance conditioning and related paradigms. When the field transitioned toward the focus on Pavlovian threat conditioning in isolation, this paved the way for the clear understanding of the psychological principles and neural and molecular mechanisms responsible for this type of learning and memory that has unfolded over recent decades. Currently, avoidance conditioning is being revisited, and with what has been learned about associative aversive learning, rapid progress is being made. We review, below, the literature on the neural substrates critical for learning in instrumental active avoidance tasks and conditioned aversive motivation.

Research on avoidance conditioning began in the late 1930s as a way to use laboratory experiments to better understand uncontrollable fear and anxiety. Avoidance was initially conceived of as a two-factor learning process in which fear is first acquired through Pavlovian aversive conditioning (so-called fear conditioning), and then behaviors that reduce the fear aroused by the Pavlovian conditioned stimulus are reinforced through instrumental conditioning. Over the years, criticisms of both the avoidance paradigm and the two-factor fear theory arose. By the mid-1980s, avoidance had fallen out of favor as an experimental model relevant to fear and anxiety. However, recent progress in understanding the neural basis of Pavlovian conditioning has stimulated a new wave of research on avoidance. This new work has fostered new insights into contributions of not only Pavlovian and instrumental learning but also habit learning, to avoidance, and has suggested that the reinforcing event underlying the instrumental phase should be conceived in terms of cellular and molecular events in specific circuits rather than in terms of vague notions of fear reduction. In our approach, defensive reactions (freezing), actions (avoidance) and habits (habitual avoidance) are viewed as being controlled by unique circuits that operate nonconsciously in the control of behavior, and that are distinct from the circuits that give rise to conscious feelings of fear and anxiety. These refinements, we suggest, overcome older criticisms, justifying the value of the new wave of research on avoidance, and offering a fresh perspective on the clinical implications of this work.

Memory formation requires the temporal coordination of molecular events and cellular processes following a learned event. During Pavlovian threat (fear) conditioning (PTC), sensory and neuromodulatory inputs converge on post-synaptic neurons within the lateral nucleus of the amygdala (LA). By activating an intracellular cascade of signaling molecules, these G-protein-coupled neuromodulatory receptors are capable of recruiting a diverse profile of plasticity-related proteins. Here we report that norepinephrine, through its actions on β-adrenergic receptors (βARs), modulates aversive memory formation following PTC through two molecularly and temporally distinct signaling mechanisms. Specifically, using behavioral pharmacology and biochemistry in adult rats, we determined that βAR activity during, but not after PTC training initiates the activation of two plasticity-related targets: AMPA receptors (AMPARs) for memory acquisition and short-term memory and extracellular regulated kinase (ERK) for consolidating the learned association into a long-term memory. These findings reveal that βAR activity during, but not following PTC sets in motion cascading molecular events for the acquisition (AMPARs) and subsequent consolidation (ERK) of learned associations.

Brain activation studies in humans have shown the dynamic nature of neuronal N‐acetylaspartate (NAA) and N‐acetylaspartylglutamate (NAAG) based on changes in their MRS signals in response to stimulation. These studies demonstrated that upon visual stimulation there was a focal increase in cerebral blood flow (CBF) and a decrease in NAA or in the total of NAA and NAAG signals in the visual cortex, and that these changes were reversed upon cessation of stimulation. In the present study we have developed an animal model in order to explore the relationships between brain stimulation, neuronal activity, CBF and NAA. We use “designer receptor exclusively activated by designer drugs” (DREADDs) technology for site‐specific neural activation, a local field potential electrophysiological method for measurement of changes in the rate of neuronal activity, functional MRS for measurement of changes in NAA and a blood oxygenation level‐dependent (BOLD) MR technique for evaluating changes in CBF. We show that stimulation of the rat prefrontal cortex using DREADDs results in the following: (i) an increase in level of neuronal activity; (ii) an increase in BOLD and (iii) a decrease in the NAA signal. These findings show for the first time the tightly coupled relationships between stimulation, neuron activity, CBF and NAA dynamics in brain, and also provide the first demonstration of the novel inverse stimulation–NAA phenomenon in an animal model.

Distinguishing threatening from nonthreatening stimuli is essential for survival and stimulus generalization is a hallmark of anxiety disorders. While auditory threat learning produces long-lasting plasticity in primary auditory cortex (Au1), it is not clear whether such Au1 plasticity regulates memory specificity or generalization. We used muscimol infusions in rats to show that discriminatory threat learning requires Au1 activity specifically during memory acquisition and retrieval, but not during consolidation. Memory specificity was similarly disrupted by infusion of PKMζ inhibitor peptide (ZIP) during memory storage. Our findings show that Au1 is required at critical memory phases and suggest that Au1 plasticity enables stimulus discrimination.

Freezing is a species-typical defensive reaction to conditioned threats. While the neural circuitry of aversive Pavlovian behavior has been extensively studied, less is known about the circuitry underlying more active responses to danger. Here we show that the flow of information between the basal amygdala (BA) and the nucleus accumbens (NAcc) is necessary for signaled active avoidance behavior. Rats trained to avoid shock by shuttling during an auditory conditioned stimulus showed increased expression of the activity-dependent protein c-Fos in the NAcc, specifically the shell subregion (NAccSh). Silencing neural activity in the NAccSh, but not in the adjacent NAcc core, disrupted avoidance behavior. Disconnection of the BA and the NAccSh was just as effective at disrupting avoidance behavior as bilateral NAccSh inactivations, suggesting learned avoidance behavior requires an intact BA-NAccSh circuit. Together, these data highlight an essential role for the amygdalar projection to the ventral striatum in aversively motivated actions.

In Europe, it is widely established that immigration increases risk for psychotic disorder. However, research has yet to confirm this association in the United States, where immigrants paradoxically report better health status than their native-born counterparts. Further, few studies have examined this topic with respect to sub-threshold psychotic experiences, which are more common than psychotic disorders in the general population. This study analyzes the (1) National Comorbidity Survey-Replication, (2) the National Latino and Asian American Survey, and (3) the National Survey of American Life, in order to determine whether generation status had any impact on risk for lifetime and 12-month PE, and whether these associations vary across racial/ethnic groups, adjusting for demographic variables and socioeconomic status. We found an absence of an immigration effect on PE across various ethnic groups and across various geographic areas, and found that immigration is actually protective among Latinos, supporting the idea that the epidemiological paradox extends to the psychosis phenotype.

A feature of multiple neuropsychiatric disorders is motor impulsivity. Recent studies have implicated serotonin (5-HT) systems in medial prefrontal cortex (mPFC) in mediating individual differences in motor impulsivity, notably the 5-HT2AR receptor (5-HT2AR) and 5-HT2CR. We investigated the hypothesis that differences in the ratio of 5-HT2AR:5-HT2CR protein expression in mPFC would predict the individual level of motor impulsivity and that the engineered loss of the 5-HT2CR would result in high motor impulsivity concomitant with elevated 5-HT2AR expression and pharmacological sensitivity to the selective 5-HT2AR antagonist M100907. High and low impulsive rats were identified in a 1-choice serial reaction time task. Native protein levels of the 5-HT2AR and the 5-HT2CR predicted the intensity of motor impulsivity and the 5-HT2AR:5-HT2CR ratio in mPFC positively correlated with levels of premature responses in individual outbred rats. The possibility that the 5-HT2AR and 5-HT2CR act in concert to control motor impulsivity is supported by the observation that high phenotypic motor impulsivity associated with a diminished mPFC synaptosomal 5-HT2AR:5-HT2CR protein:protein interaction. Knockdown of mPFC 5-HT2CR resulted in increased motor impulsivity and triggered a functional disruption of the local 5-HT2AR:5-HT2CR balance as evidenced by a compensatory upregulation of 5-HT2AR protein expression and a leftward shift in the potency of M100907 to suppress impulsive behavior. We infer that there is an interactive relationship between the mPFC 5-HT2AR and 5-HT2CR, and that a 5-HT2AR:5-HT2CR imbalance may be a functionally relevant mechanism underlying motor impulsivity.

Pavlovian threat conditioning is a behavioral paradigm that has been successfully utilized to define the mechanisms underlying threat (fear) memory formation. The amygdala is a temporal lobe structure required for the acquisition, consolidation, and expression of threat (fear) memories. In particular, the lateral nucleus of the amygdala (LA) is the major input structure of the amygdala and is required for all aspects of threat learning and memory. The LA expresses many neurotransmitter and neuromodulator receptors. This chapter covers the molecular mechanisms that occur downstream of these receptors and how they influence LA-dependent Pavlovian threat learning.