Targeting corticotropin-releasing hormone receptor type 1 (Crhr1) neurons: validating the specificity of a novel transgenic Crhr1-FlpO mouse.

Abstract

Corticotropin-releasing hormone (CRH) signaling through its cognate receptors, CRHR1 and CRHR2, contributes to diverse stress-related functions in the mammalian brain. Whereas CRHR2 is predominantly expressed in choroid plexus and blood vessels, CRHR1 is abundantly expressed in neurons in discrete brain regions, including the neocortex, hippocampus and nucleus accumbens. Activation of CRHR1 influences motivated behaviors, emotional states, and learning and memory. However, it is unknown whether alterations in CRHR1 signaling contribute to aberrant motivated behaviors observed, for example, in stressful contexts. These questions require tools to manipulate CRHR1 selectively. Here we describe and validate a novel Crhr1-FlpO mouse. Using bacterial artificial chromosome (BAC) transgenesis, we engineered a transgenic mouse that expresses FlpO recombinase in CRHR1-expressing cells. We used two independent methods to assess the specificity of FlpO to CRHR1-expressing cells. First, we injected Crhr1-FlpO mice with Flp-dependent viruses expressing fluorescent reporter molecules. Additionally, we crossed the Crhr1-FlpO mouse with a transgenic Flp-dependent reporter mouse. CRHR1 and reporter molecules were identified using immunocytochemistry and visualized via confocal microscopy in several brain regions in which CRHR1 expression and function is established. Expression of Flp-dependent viral constructs was highly specific to CRHR1-expressing cells in all regions examined (over 90% co-localization). In accord, robust and specific expression of the Flp-dependent transgenic reporter was observed in a reporter mouse, recapitulating endogenous CRHR1 expression. The Crhr1-FlpO mouse enables selective genetic access to CRHR1-expressing cells within the mouse brain. When combined with Cre-lox or site-specific recombinases, the mouse facilitates intersectional manipulations of CRHR1-expressing neurons.