Cellular, circuit and transcriptional framework for modulation of itch in the central amygdala
- Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, United States
- Medical Scientist Training Program, Washington University School of Medicine, United States
- Neuroscience Program, Washington University School of Medicine, United States
- Department of Pathology & Immunology, Washington University School of Medicine, United States
- Genome Technology Access Center, Washington University School of Medicine, United States
- Department of Biomedical Engineering, University of Washington, United States
- Departments of Anesthesiology and Pharmacology, University of Washington, United States
- Departmentsof Neuroscience and Biomedical Engineering, Washington University School of Medicine, United States
Figures
Figure 1 with 1 supplement
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 …
Figure 1—figure supplement 1
Anatomical location of the GCaMP6s-expressing central amygdala (CeA) neurons and fiber placements for imaging activity during itch behaviors.
(a) Scheme demonstrating viral injection strategy and fiber placement to record CeA Vgat neural activity in response to chloroquine. (b) Representative image of the CeA of Vgat Cre mice in which …
Figure 2 with 4 supplements
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 …
Figure 2—figure supplement 1
Anatomical location of cFos-expressing (itch-activated) neurons in the central amygdala (CeA) following chloroquine injection in the nape of the neck.
(a) Representative sections showing cFos labeling at low and high magnification following chloroquine injection in the nape of the neck. Scale bar 250 μm in left panel, 50 μm in the right panel. (b) …
Figure 2—figure supplement 2
Optogenetic reactivation of itch-TRAPed neurons in the left central amygdala (CeA) neurons promotes scratching.
(a) Viral strategy to selectively express ChR2 or eYFP in chloroquine-activated neurons of the left CeA. Experimental timeline to FosTRAP ChR2/eYFP in CeA. (b) Viral strategy to selectively express …
Figure 2—figure supplement 3
Chemogenetic activation of the central amygdala (CeA) neurons promotes itch behaviors.
(a) Viral strategy to selectively express excitatory DREADDs in chloroquine-activated CeA neurons. (b) Experimental timeline to FosTRAP DREADDs in CeA. (c) Representative section showing CeA …
Figure 2—figure supplement 4
Chemogenetic manipulation of FosTRAPed central amygdala (CeA) neurons modulates nociceptive behaviors.
(a) Viral strategy to selectively express excitatory and inhibitory DREADDs in itch-activated CeA neurons. (b) Chemogenetic activation of chloroquine-TRAPed CeA neurons does not significantly alter …
Figure 3 with 3 supplements
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) …
Figure 3—figure supplement 1
Optogenetic and chemogenetic activation of FosTRAPed central amygdala (CeA) neurons causes anxiety but no freezing, whereas inhibition of these neurons does not affect anxiety state.
(a) Viral strategy to selectively express various constructs in chloroquine-activated CeA neurons. (b) Chemogenetic activation of chloroquine-TRAPed CeA neurons significantly reduces time spent in …
Figure 3—figure supplement 2
Chemogenetic manipulation of FosTRAPed central amygdala (CeA) neurons does not affect feeding behaviors.
(a, b) Viral constructs used and schematic of experimental timeline. Chemogenetic activation (c) or inhibition (d) of chloroquine-TRAPed CeA neurons has no effect on food intake. n = 6–9 per group. …
Figure 3—figure supplement 3
Chemogenetic manipulation of FosTRAPed central amygdala (CeA) neurons does not affect reward-seeking behaviors.
(a) Viral strategy to selectively express excitatory and inhibitory DREADDs in itch-activated CeA neurons. (b) Experimental timeline (FR1 = fixed ratio 1; FR3 = fixed ratio 3; PR = progressive …
Figure 4
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 …
Figure 5 with 1 supplement
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. …
Figure 5—figure supplement 1
Anatomical tracing to identify connections between the central amygdala (CeA) and the periaqueductal gray (PAG).
(a) Retrograde tracer CTB 488 injection into the ventral periaqueductal gray (vPAG) to confirm projections from CeA in C57BL/6J mice. Representative image of vPAG showing CTB488 injection. (b) CeA …
Figure 6 with 2 supplements
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 …
Figure 6—figure supplement 1
Fluorescence-activated cell sorting (FACS) of central amygdala (CeA) FosTRAPed neurons to perform RNA-seq and transcriptional analysis of chloroquine-TRAPed CeA neurons.
(a) Schematic showing dissociation and FACS of CeA FosTRAPed neurons. (b, d) Labeling of dissociated neurons with Neurotrace reveals a clear subset of events that can be found in the bottom of the …
Figure 6—figure supplement 2
Transcriptional analysis of central amygdala (CeA) FosTRAPed neurons.
Total (a) mapped and (b) unique mapped reads per million are similar between tdTomato+ve and tdTomato-ve neurons. t test, t = 0.07, df = 4, p=0.94 for mapped reads and t = 0.08, df = 4, p=0.93 for …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Species (Mus musculus), strain | Ai9-tdTomato mice (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) | The Jackson Laboratory | 007909 | Ai9 |
| Species (Mus musculus), strain | FosCreERT2 mice (B6.129(Cg)-Fostm1.1(cre/ERT2)Luo/J) | The Jackson Laboratory | 21882 | FosCreER |
| Species (Mus musculus), strain | Vgat-ires-Cre (Slc32a1tm2Lowl) | The Jackson Laboratory | 028862 | Vgat Cre |
| Species (Mus musculus), strain | Vglut2-ires-Cre (Slc17a6tm2) | The Jackson Laboratory | 028863 | Vglut2 Cre |
| Species (Mus musculus), strain | C57BL\6J | In bred | NA | NA |
| Recombinant DNA reagent | rAAV5/hSyn-DIO-hM3Dq-mCherry | University of North Carolina Vector Core | NA | 75 nL of virus |
| Recombinant DNA reagent | rAAV5/hSyn-DIO-hM4Di-mCherry | University of North Carolina Vector Core | NA | 75 nL of virus |
| Recombinant DNA reagent | rAAV5-DIO-ChR2-eYFP | University of North Carolina Vector Core | NA | 100 nL of virus |
| Recombinant DNA reagent | rAAV5/hSyn-DIO-mCherry | University of North Carolina Vector Core | NA | 75 nL of virus |
| Recombinant DNA reagent | rAAV5-DIO-eYFP | University of North Carolina Vector Core | NA | 100 nL of virus |
| Recombinant DNA reagent | rAAV5/EF1α-FLEX-TVAmCherry | University of North Carolina Vector Core | NA | 75 nL of virus (1:1 with RG) |
| Recombinant DNA reagent | rAAV5/CAG-FLEX-RG | University of North Carolina Vector Core | NA | 75 nL of virus (1:1 with TVA) |
| Recombinant DNA reagent | EnvA G-deleted Rabies-GFP | University of North Carolina Vector Core | NA | 100 nL of virus |
| Chemical compound, drug | Clozapine-N-oxide (CNO) | Enzo Life Sciences | BML-NS105 | NA |
| Chemical compound, drug | 4-Hydroxytamoxifen | Sigma–Aldrich | H6278-10MG | NA |
| Chemical compound, drug | Chloroquine | Sigma–Aldrich | C6628 | NA |
| Antibody | Rb-mCherry | Clontech | Cat. #: 632543 | 1:1000, RRID:AB_2307319 |
| Antibody | Ch-GFP | AVES | A11122 | 1:2000, AB_10000240 |
| Antibody | Rb-cFos | Cell Signaling | Cat. #: D82C12 | 1:1000, RRID:AB_10557109 |
| Sequence-based reagent (smFISH) | mm-Fos | Advanced Cell Diagnostics | 316921 | NA |
| Sequence-based reagent (smFISH) | mm- Ntsr2 | Advanced Cell Diagnostics | 452311 | NA |
| Sequence-based reagent (smFISH) | mm- GPR88 | Advanced Cell Diagnostics | 317451 | NA |
| Sequence-based reagent (smFISH) | mm-Penk | Advanced Cell Diagnostics | 318761 | NA |
| Sequence-based reagent (smFISH) | mm-Gabarg1 | Advanced Cell Diagnostics | 501401 | NA |
| Sequence-based reagent (smFISH) | mm-Oprm1 | Advanced Cell Diagnostics | 315841 | NA |
| Sequence-based reagent (smFISH) | mm-Chrm1 | Advanced Cell Diagnostics | 495291 | NA |
| Software, algorithm | Ethovision XT | Noldus | https://www.noldus.com/ethovision-xt | NA |
| Software, algorithm | Prism7 | GraphPad | https://identifiers.org/RRID/RRID:SCR_002798 | NA |
| Software, algorithm | MATLAB, 2018b | MathWorks | https://www.mathworks.com/products/matlab.html | NA |
| Software, algorithm | RZ5P | Tucker-Davis Technologies | https://www.tdt.com/system/fiber-photometry-system/ | NA |
