Viral Retrograde Tracing of CRH-Expressing Axonal Projections to the Dorsal Striatum

Abstract

Stress is the body's physiological and psychological response to perceived threats, mediated by the Hypothalamic-Pituitary-Adrenal (HPA) axis and initiated by the release of Corticotropin-releasing Hormone (CRH) (Herman et al., 2016). Under acute conditions, stress can help the body adapt to its environment by activating the “fight or flight” response (Leonard & Myint, 2009), but chronic stress can cause impaired reward-based learning and reinforced habitual behavior (Giovanniello et al., 2025). These effects of chronic stress may be partly mediated through altered CRH signaling in the dorsal striatum, a region implicated in decision-making through prediction error-based learning from unexpected rewards and punishments (Balleine et al., 2007). Although the dorsal striatum expresses CRH receptors, it lacks local CRH-producing neurons (Horváth et al., 2023), suggesting that axons releasing CRH onto the dorsal striatum project from other regions, though these have not been characterized. To identify sources of CRH+ projections to the dorsal striatum, we injected a Cre-dependent retrograde virus into the dorsal striatum of transgenic CRH-Cre mice, selectively labeling CRH+ neurons with a fluorescent reporter. Brain sections were imaged using fluorescence microscopy and CRH+ cell counts were quantified by region using automated cell detection software. Results revealed unexpectedly high CRH+ labeling in motor and somatosensory cortices, likely reflecting limitations related to viral spread or off-target cell detection. Moderate CRH+ projections from the basolateral amygdala, anterior cingulate area, and prelimbic cortex were also observed, identifying stress-related CRH sources in the limbic system as candidates for CRH+ input to the dorsal striatum for examination in future studies.