Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior
- Department of Physiology, Louisiana State University Health Sciences Center, United States
- Department of Pharmacology, University of North Carolina, United States
- Institute of Molecular Medicine, University of Texas Health Sciences Center, United States
- Bowles Center for Alcohol Studies, University of North Carolina, United States
- Department of Integrative Biology and Pharmacology, McGovern Medical School at UT Health, United States
- Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, United States
- Alcohol & Drug Abuse Center of Excellence, Louisiana State University Health Sciences Center, United States
- Southeast Louisiana VA Healthcare System (SLVHCS), United States
Figures
Figure 1
Design of the Crhr1-Cre2aTom BAC transgene.
(A)Crhr1 is located on chromosome 10 in the rat. A BAC clone containing 196 kb of DNA surrounding the Crhr1 coding region includes 100 kb of upstream and 80 kb of downstream DNA, where the majority …
Figure 2
Validation of transgenic expression of iCre/tdTomato in CRF1+ expressing neurons located in the medial central nucleus of the amygdala (CeAm).
(A) A low-magnification image of a section containing the amygdala from a CRF1-Cre rat, immunohistochemically labeled for tdTomato. Expression of the CRF1-Cre transgene is broadly very similar to …
Figure 3
CRF1-driven expression of Cre/tdTomato in the CeA.
(A) Visualization of CRF using immunofluorescent labeling (green) in a rat carrying the CRF1-Cre2aTom transgene (red) reveals minimal cellular expression of CRF1 in the lateral central nucleus of …
Figure 4
Basal membrane properties and inhibitory synaptic transmission in CeAm CRF1-Cre-tdTomato neurons.
(A) Basal membrane properties (Membrane Capacitance, Cm; Membrane Resistance, Rm; Decay Constant, Tau; Membrane Potential, Vm) from male and female CRF1+ CeAm neurons. (B) Representative …
Figure 5
Spontaneous firing activity and CRF sensitivity of CeAm CRF1-Cre-tdTomato neurons.
(A) Representative cell-attached recording of spontaneous firing activity in a CRF1+ CeAm neuron from a male CRF1-Cre-tdTomato rat before and during CRF (200 nM) application. (B) Representative …
Figure 6
Validation of DREADD expression and function in CeA CRF1-Cre-tdTomato neurons.
(A) Representative images of CRF1-Cre-tdTomato cells (red) and c-Fos immunostaining (white) in CeAm of rats that were given intra-CeA microinjections of AAV8-hSyn-DIO-HA-hM3D(Gq)-IRES-mCitrine …
Figure 7
Effects of chemogenetic stimulation of CeA CRF1-Cre-tdTomato neurons on nociception and anxiety-like behaviors.
(A) Representative image of AAV8-hSyn-DIO-HA-hM3D(Gq)-IRES-mCitrine expression (green) in the CeA. Scale bar: 500 µm. BLA: basolateral amygdala, Opt: optic tract. (B) Timeline of experimental …
Figure 8
Crhr1 and iCre mRNA expression in BLA of CRF1-Cre-tdTomato rats.
(A) Crhr1 (green) and iCre (red) mRNA are highly co-expressed in BLA and surrounding areas. (B) Within the BLA, 98.5% of Crhr1+ + co-express iCre (top), and 99.4% of iCre+ + co-express Crhr1 (bottom).
Figure 9
Fluorescent images of brainwide tdTomato expression.
(A) A schematic summary of brain areas that were surveyed for tdTomato expression. Solid red circles indicate brain areas that contain tdTomato cells and dashed circles indicate brain areas that …
Tables
Table 1
Comparison of CRF1-Cre-tdTomato cell densities with Crhr1 mRNA expression levels in rats and CRF1-GFP cell densities in mice in several areas across the rostrocaudal axis of the brain.
+: Some cells are positive, ++: A substantial number of cells are positive, +++: Most cells are positive.
| Brain area | Density of tdTomato cells in CRF1-Cre-tdTomato rats | Expression level of Crhr1 mRNA in wildtype rats (Van Pett et al., 2000) | Density of GFP cells in CRF1-GFP Mice (Justice et al., 2008) |
|---|---|---|---|
| PrL | ++ | ++ | ++ |
| IL | ++ | ++ | ++ |
| BNST | +++ | +++ | +/++ |
| LSv | + | + | - |
| PVN | + | + | ++ |
| PIR | +++ | +++ | ++/+++ |
| MeA | ++ | + | +/++ |
| LHA | + | + | ++ |
| Hipp | + (subiculum, CA3)/+++ (CA1) | ++ (CA1, CA3, subiculum) | + (subiculum, CA3)/+++ (CA1) |
| PVT | ++ | ++ | - |
| LHb | - | - | ++ |
| MHb | + | - | - |
| VTA | ++ | ++ | ++ |
| PAG | ++ | + | + |
| DR | ++ | + | ++ |
| LC | - | - | +/- |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Rattus norvegicus) | BAC containing the Crfr1 genomic locus | Riken Gene Engineering Division | RNB2-336H12 | |
| Transfected construct (Rattus norvegicus) | iCre-2a-tdTomato BAC transgene | This paper | See Results, Design of CRF1-Cre BAC; Contact Justice Lab | |
| Genetic reagent (Rattus norvegicus) | CRF1-Cre-tdTomato rat | This paper | See Results, Generation of Transgenic Rats; Contact Gilpin Lab | |
| Recombinant DNA reagent | AAV8-hSyn-DIO-HA-hM3D(Gq)-IRES-mCitrine | Addgene | Cat# 50454-AAV8 | |
| Recombinant DNA reagent | AAV5-hSyn-DIO-EGFP | Addgene | Cat# 50457-AAV5 | |
| Antibody | Anti-c-Fos (Rabbit polyclonal) | Abcam | Cat# ab190289, RRID:AB_2737414 | (1:1000) |
| Antibody | Anti-HA-Tag (Rabbit monoclonal) | Cell Signaling | Cat# 3724, RRID:AB_1549585 | (1:250) |
| Antibody | Anti-RFP (Rabbit monoclonal) | Abcam | Cat# ab34771, RRID:AB_777699 | (1:500) |
| Antibody | Anti-CRF (Rabbit monoclonal) | The Salk Institute | Rc-68 | (1:2000) |
| Commercial assay or kit | RNAscope Multiplex Fluorescent Kit v2 | ACD Bio | iCre and Crfr1 probes | |
| Commercial assay or kit | TSA Detection Kit | Akoya Biosciences | Cat# NEL701A001KT | |
| Commercial assay or kit | Prolong Gold Antifade Reagent with DAPI | Invitrogen | Cat# P36935 | |
| Chemical compound, drug | Clozapine-n-oxide | NIH Drug Supply Program | ||
| Chemical compound, drug | Corticotropin-releasing factor | Tocris | Cat# 1,607 | |
| Software, algorithm | Mini Analysis | Synaptosoft Inc. | RRID:SCR_002184 | |
| Software, algorithm | Clampfit 10.6 | Molecular Devices | ||
| Software, algorithm | Prism 7.0 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | SPSS 25 | IBM SPSS | RRID:SCR_019096 |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67822/elife-67822-transrepform1-v2.docx
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Source data 1
Slice electrophysiology data.
- https://cdn.elifesciences.org/articles/67822/elife-67822-data1-v2.xlsx
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Source data 2
Behavioral data.
- https://cdn.elifesciences.org/articles/67822/elife-67822-data2-v2.xlsx
