Identification of brain target neurons using a fluorescent conjugate of corticotropin-releasing factor

Corticotropin-releasing factor (CRF) is a peptide well known for its role in coordinating various neuroendocrine, autonomic, and behavioral components of the vertebrate stress response, including rapid enhancement of locomotor activity. Although CRF's locomotor enhancing properties are well documented, the neuronal mechanisms and specific target neurons that underlie the peptide's effect on locomotor behavior remain poorly understood. In the present study, we describe the synthesis and functional characteristics of a CRF rhodamine analogue TAMRA-X conjugate mixture (CRF-TAMRA 1), to be used for tracking this peptide's internalization into target neurons in the brainstem of an amphibian, the roughskin newt (Taricha granulosa). CRF-TAMRA 1 conjugate administration into the lateral cerebral ventricle resulted in a rapid, endosomal-like internalization of fluorescence into brainstem medullary neurons. In addition, central CRF-TAMRA 1 administration produced neurobehavioral effects comparable to the native peptide, effects that were blocked by pre-treatment with the CRF receptor antagonist, alpha-helical CRF. Taken together, our results show the efficacy of CRF-TAMRA 1 as a novel tool for tracking CRF internalization into targets neurons in vivo and ultimately, aiding in elucidating the neuronal mechanisms and circuitry underlying CRF's influence on behavioral and physiological responses to stress.