Generation of a CRF 1 -Cre transgenic rat and the role of central amygdala CRF 1 cells in 1 nociception and anxiety-like behavior

Corticotropin-releasing factor type-1 (CRF 1 ) receptors are critical to stress responses because 26 they allow neurons to respond to CRF released in response to stress. Our understanding of the 27 precise role of CRF 1 -expressing neuronal populations in CRF-mediated behaviors has been 28 largely limited to mouse experiments due to the lack of genetic tools available to selectively 29 visualize and manipulate CRF 1+ cells in rats. Here, we describe the generation and validation of 30 a transgenic CRF 1 -Cre- td Tomato rat, which expresses a bicistronic iCre-2A- td Tomato transgene 31 directed by 200kb of promoter and enhancer sequence surrounding the Crhr1 cDNA present 32 within a BAC clone, that has been transgenically inserted into the rat genome. We report that 33 Crhr1 and Cre mRNA expression are highly colocalized in CRF 1 -Cre- td Tomato rats within both 34 the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral 35 amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, 36 inhibitory synaptic transmission, and responses to CRF bath application in td Tomato + neurons 37 are similar to those previously reported in GFP + cells in CRFR1-GFP mice. We show that 38 stimulatory DREADD receptors can be selectively targeted to CeA CRF 1+ cells via virally 39 delivered Cre-dependent transgenes, that transfected Cre/ td Tomato + cells are activated by 40 clozapine-n-oxide in vitro and in vivo , and that activation of these cells in vivo increases anxiety- 41 like behavior and nocifensive responses. Outside the amygdala, we show that Cre- td Tomato is 42 expressed in several brain areas across the rostrocaudal axis of the CRF 1 -Cre- td Tomato rat 43 brain, and that the expression pattern of Cre- td Tomato cells is similar to the known expression 44 pattern of CRF 1 cells. Given the accuracy of expression in the CRF 1 -Cre rat, modern genetic 45 techniques used to investigate the anatomy, physiology, and Here, we describe the generation of a novel CRF 1 -Cre- td Tomato transgenic rat line. We show that Crhr1 and iCre mRNA are expressed in the same neurons in the CeA and BLA using hybridization histochemistry. In the CeA, we show that td Tomato + neurons are abundant in the CeAm, and that they are surrounded and in contact with CRF-containing puncta. We recorded membrane properties, inhibitory synaptic transmission, and spontaneous firing in CeA CRF 1 - Cre- td Tomato cells and show that these cells are sensitive to CRF. In addition, we show that Cre-dependent DREADD receptors can be targeted for expression by CeA CRF 1 cells such that DREADD stimulation of CRF 1+ CeA neurons increases nociception and anxiety-like behaviors, recapitulating prior work using pharmacological strategies. Outside the amygdala, we analyzed the expression of CRF 1 -Cre- td Tomato cells in several brain areas across the rostrocaudal axis and show that these cells are found in brain areas known to contain CRF 1 -expressing cells. These anatomical, electrophysiological and functional data support the utility of this CRF 1 -Cre- td Tomato transgenic rat line for the study of CRF 1 neural circuit function, and will be an important new resource that will complement CRFR1:GFP and CRFR1:Cre mice 2008; 111 Jiang Sanford and CRF-Cre rats (Pomrenze 2015) for the study of CRF signaling in physiology and behavior. inhibitory synaptic transmission, and validated the sensitivity of td 1+ cells in male and female rats. In addition, we showed that stimulatory receptors can be targeted to CeA 1 -Cre- td Tomato cells using a Cre-dependent expression strategy, that systemic CNO treatment induces c-Fos in hM3D(Gq)-transduced CRF 1+ cells in CeA, and that CNO induces membrane depolarization and spontaneous firing activity in hM3D(Gq)-transduced CRF 1+ cells in CeA. We showed that hM3D(Gq)-mediated stimulation of CeA CRF 1+ cells increases anxiety-like behavior, as measured by EPM and open field tests, as well as mechanical nociception as measured by the Von Frey test. Finally, we report that Crhr1 and iCre mRNA expression are highly colocalized in the BLA, and that Cre- td Tomato is 341 expressed in several brain areas across the rostrocaudal axis of the rat brain in expected patterns. Collectively, this work provides cellular, electrophysiological, and behavioral data demonstrating the validity and reliability of a new transgenic rat model for the identification and selective manipulation of CRF 1+ neurons in the CeA.