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 precise role of CRF1-expressing neuronal populations 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, which expresses a bicistronic iCre-2A-tdTomato transgene directed by 200kb of promoter and enhancer sequence surrounding the Crhr1 cDNA present within a BAC clone, that has been transgenically inserted into the rat genome. We report that Crhr1 and Cre mRNA expression are highly colocalized in CRF1-Cre-tdTomato rats within 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 selectively 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 behavior and nocifensive responses. Outside the amygdala, we show that Cre-tdTomato is expressed in several brain areas across the rostrocaudal axis of the CRF1-Cre-tdTomato rat 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 and circuits can now be performed in assays that require the use of rats as the model organism.

Psychedelic compounds directly excite 5-HT2ALayer 5 Pyramidal Neurons in the Prefrontal Cortex through a 5-HT2AGq -mediated activation mechanism

“…Series resistance was continuously monitored with a hyperpolarizing 10mV pulse; cells with axis resistance >20 MΩ were excluded from the data set. 5HT 2A R expressing neurons were identified via Tdtomato expression using fluorescent optics and brief (<2s) episodic illumination as previously described 37 . During voltage clamp recording (V hold = -70mV), electrophysiological properties of cells were determined by pClamp 10 Clampex software using a 10mV pulse delivered after breaking into the cell.…”

Chronic Alcohol Drinking Drives Sex-Specific Differences in Affective Behavior and Medial Prefrontal Cortex Activity in CRF1:Cre:Tdtomato Transgenic Rats

“…Consistent with some, but not all reports, female rats were more sensitive to mechanical stimuli (measured by Von Frey)(Araya et al, 2020; Gogulski and Craft, 2022; Mapplebeck et al, 2018) but these sex differences were specific to mechanical sensitivity. Pain sensitivity in male and female CRF1:cre: td Tomato rats was previously examined, however, the study was not appropriately powered to detect sex effects(Weera et al, 2022). In contrast to our previous work, we did not see any basal sex differences in either the splash or NSF test(Quadir et al, 2022).…”

“…EtOH Alters Affect & PFC Activity in CRF1-cre rats 2. METHODS 2.1 Subjects 32 adult male and female (n=16/sex) CRF1:cre: td Tomato rats 52 (8)(9)(10)(11) weeks old, bred in-house) were housed in temperature and humidity-controlled rooms with 12h light/dark cycle (7:00 am lights ON). Rats had ad libitum access to food and water unless otherwise stated.…”

“…The prefrontal cortex and amygdala are two regions extensively involved in both affective behavior and AUDs(Abernathy et al, 2010; Agoglia and Herman, 2018; Centanni et al, 2019; Egli et al, 2012; Hare and Duman, 2020; Jacobs and Moghaddam, 2021; Kenwood et al, 2022; Lu and Richardson, 2014; McCool, 2021; Melkumyan and Silberman, 2022; Neugebauer et al, 2020; Roberto et al, 2021; Smith and Torregrossa, 2021; Veinante et al, 2013; Zhang et al, 2021). Both regions have an abundance of CRF1-containing cells (Agoglia et al, 2020; Calakos et al, 2017; Conte et al, 2019; Gilpin et al, 2015; Patel et al, 2022; Robinson et al, 2019; Weera et al, 2022). However, the specific contributions of these cells to AUD and associated negative affect remains unclear.…”

“…Indeed, alcohol preferring rats have increased CRF1 levels in the cingulate cortex, motor cortex, and somatosensory cortex 49 . CRF1 is also abundantly expressed in the medial prefrontal cortex, 50, 51 but there are limited studies examining the effects of chronic EtOH drinking on prefrontal CRF1-containing neurons.…”