Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior

Abstract

Corticotropin-releasing factor type-1 (CRF₁) receptors are critical to stress responses because they allow neurons to respond to CRF released in response to stress. Our understanding of the role of CRF₁-expressing neurons in CRF-mediated behaviors has been largely limited to mouse experiments due to the lack of genetic tools available to selectively visualize and manipulate CRF₁⁺ cells in rats. Here, we describe the generation and validation of a transgenic CRF₁-Cre-^tdTomato rat. We report that Crhr1 and Cre mRNA expression are highly colocalized in both the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, inhibitory synaptic transmission, and responses to CRF bath application in ^tdTomato⁺ neurons are similar to those previously reported in GFP⁺ cells in CRFR1-GFP mice. We show that stimulatory DREADD receptors can be targeted to CeA CRF₁⁺ cells via virally delivered Cre-dependent transgenes, that transfected Cre/^tdTomato⁺ cells are activated by clozapine-n-oxide in vitro and in vivo, and that activation of these cells in vivo increases anxiety-like and nocifensive behaviors. Outside the amygdala, we show that Cre-^tdTomato is expressed in several brain areas across the brain, and that the expression pattern of Cre-^tdTomato cells is similar to the known expression pattern of CRF₁ cells. Given the accuracy of expression in the CRF₁-Cre rat, modern genetic techniques used to investigate the anatomy, physiology, and behavioral function of CRF₁⁺ neurons can now be performed in assays that require the use of rats as the model organism.

Keywords: CRF; anxiety; central amygdala; crf1 receptor; neuroscience; nociception; rat.

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 of promoter and enhancer sequences that control Crhr1 expression are located, was obtained (Riken, RNB2-336H12). There are no other sequences within this 196 kb DNA clone have been annotated as coding sequences for genes other than Crhr1. (B) A transgene containing 5’ (blue) and 3’ (magenta) targeting sequences, a bicistronic iCre 2 A fused tdTomato (red) sequence, 3’ polyA/WPRE stabilizing sequence, and a F3 flanked neomycin resistance sequence (yellow) was constructed then transformed into E. coli containing the RNB2-336H12 BAC construct. (C) Using recombineering techniques we isolated BAC clones in which targeted insertion of the transgene at the translation start site of Crhr1 (ATG) was confirmed by PCR/sequencing. A single Bacterial clone containing the transgene inserted BAC was sent to the UNC transgenic facility where BAC DNA was purified and injected into single cell, fertilized rat oocytes. Two independent rat lines were recovered in which the entire BAC sequence (confirmed by PCR) was inserted into genomic DNA, of which one line displayed transgenic expression in a pattern representative of known Crhr1 expression patterns.

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 previous reports of CRF₁ expression in both rat and mouse. (B) Within the boxed region of panel A, higher magnification reveals CRF₁⁺ cells in the lateral amygdala (LA), basolateral amygdala (BLA), medial portion central amygdala (CeAm), and medial amygdala (MeA). The lack of significant labeling in the CeAl is consistent with reports using both in situ hybridization and transgenic reporters to detect CRF₁ expression. (C) Micrograph of the CeA from the region boxed in panel B allows visualization of mRNA encoding iCre (red) and Crhr1 (green) along with nuclei stained with DAPI (blue). (D–G) Higher magnification images of the boxed region in (C) allows visualization of mRNA for Crhr1 (D, green), and iCre (E, red), with nuclei visualized by DAPI staining (F). (G) Merged images reveals that many CeAm neurons that are positive for Crhr1 mRNA are also positive for iCre mRNA (arrows point to double positive neurons). Quantification of coincidence of in situ hybridization for both Crhr1 and iCre mRNAs demonstrates that >90% of Crhr1-positive cells are also positive for iCre in the CeAm (n = 3). (H) Graphical representation of quantification of coincident labeling, or (I) a table of the precise counts from each of three male and three female CRF₁-Cre transgenic animals. We observed greater than 90% of neurons positive for both Crhr1 and iCre in the CeAm.

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 the amygdala (CeAl) where CRF is highly abundant. This discrepancy in CRF localization compared to CRF₁ expression is consistent with previous reports of CRF₁ expression in both rat and mouse. In contrast to the CeAl, the medial central nucleus of the amygdala (CeAm) contains many CRF₁⁺ neurons (reported by the CRF₁-Cre2Atom transgene), in contact with puncta positive for CRF peptide. (B) High-resolution images from the boxed regions of CeAl and CeAm in panel A. CRF staining is dense in both the CeAl and CeAm (top panels); however, cellular expression of the CRF₁-Cre2aTom transgene is low in the CeAl, while many neurons in the CeAm are positive for CRF₁ expression (middle panels). Merged images (lower panels) display the coincident staining of CRF₁⁺neurons with CRF puncta in the CeAm, suggesting that stress driven CRF release directly signals to CRF₁⁺ neurons in the CeAm to modulate neural excitability to influence the output of CeAm neurons. LA – lateral amygdala, BLA – basolateral amygdala.

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 current-evoked spiking properties from male (top) and female (bottom) CRF₁⁺ CeAm neurons. (C) Basal spontaneous inhibitory postsynaptic currents (sIPSCs) from male (top) and female (bottom) CRF₁⁺ CeAm neurons (right). Inset: representative fluorescent (left) and infrared differential interference contract (IR-DIC, right) image of a CRF₁⁺ CeAm neuron targeted for recording. Scale bar = 20 μm. (D) Average sIPSC frequency (left) and sIPSC amplitude (right) from male and female CRF₁⁺ CeAm neurons. Raw data are available in Source Data File 1.

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 cell-attached recording of spontaneous firing activity in a CRF₁⁺ CeAm neuron from a female CRF₁-Cre-^tdTomato rat before and during CRF (200 nM) application. (C) Summary of changes in spontaneous firing activity with CRF application in CRF₁⁺ CeAm neurons from male CRF₁-Cre-^tdTomato rats.*p < 0.05 by paired t-test. (D) Summary of changes in spontaneous firing activity with CRF application in CRF₁⁺ CeAm neurons from female CRF₁-Cre-^tdTomato rats.*p < 0.05 by paired t-test. (E) Normalized change in firing activity in CRF₁⁺ CeAm neurons from male and female CRF₁-Cre-^tdTomato rats.*p < 0.05 by one-sample t-test. Raw data are available in Source Data File 1.

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 (active virus) or AAV5-hSyn-DIO-EGFP (control virus). Scale bar: 50 µm. (B) CNO treatment 90 min before sacrifice increased the percentage of c-Fos^{+ td}Tomato cells in CeAm of rats that were given active virus compared to rats that were given control virus microinjections. *p < 0.05. (C) Representative whole-cell current clamp recording of membrane potential and firing activity in a CRF₁⁺ CeAm neuron from a male CRF₁-Cre-^tdTomato rat before and during CNO (10 μM) application. (D) Summary of the change in membrane potential (left) and action potentials (right) in male CRF₁⁺ CeAm neurons after CNO application. *p < 0.05 by paired t-test. (E) Representative whole-cell current clamp recording of membrane potential and firing activity in a CRF₁⁺ CeAm neuron from a female CRF₁-Cre-^tdTomato rat before and during CNO (10 μM) application. (D) Summary of the change in membrane potential (left) and action potentials (right) in female CRF₁⁺ CeAm neurons after CNO application. *p < 0.05 by paired t-test. Raw data are available in Source Data File 1.

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 procedures. (C) CNO treatment decreased paw withdrawal thresholds in the Von Frey test of mechanical nociception in rats that were given intra-CeA hM3D(Gq) virus microinjections. There were no effects of treatment on paw withdrawal thresholds in the EGFP control group. (D) CNO treatment had no effects on paw withdrawal latencies in either the hM3D(Gq) or EGFP groups in the Hargreaves test of thermal nociception. (E) CNO treatment decreased the percent time spent in open arms in the EPM test in the hM3D(Gq) group, but had no effect in the EGFP group. (F) CNO treatment decreased the percent time spent in the center of the arena in the OF test in the hM3D(Gq) group, but had no effect in the EGFP group. (G) CNO treatment had no effect on percent time spent in the light box in the LD test. *p < 0.05. Raw data are available in Source Data File 2. iCre and Crhr1 mRNA are highly co-expressed in BLA.

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 were devoid of ^tdTomato cells. (B–X) Low (4 x) and high-magnification (×20 ) images of tdTomato (red) and DAPI (blue) fluorescent signals. Yellow boxes demarcate areas from which high-magnification (×20 ) images were acquired. (B) 4 x image of the PrL, IL, and surrounding landmarks. (C) 20 x image of PrL and IL. (D) 4 x of BNST and LSv. (E) 20 x image of BNST and (F) LSv. (G) 4 x and (H) 20 x image of PVN. (I) 4 x image of PIR, MeA, and LHA. (J) 20 x image of PIR, (K) MeA, and (L) LHA. (M) 4 x and (N) 20 x image of hippocampus. (O) 4 x image PVT, LHB, and MHb. (P) 20 x image of PVT and (Q) LHb/MHb. (R) 4 x and (S) 20 x image of VTA. (T) 4 x image of PAG and DR. (U) 20 x image of PAG and (V) DR. (W) 4 x and (X) 20 x image of LC. 3 V: 3rd ventricle, 4 V: 4th ventricle, D3V: dorsal 3rd ventricle, Aq: cerebral aqueduct.