Agonist-induced internalization of corticotropin-releasing factor receptors in noradrenergic neurons of the rat locus coeruleus

Abstract

Corticotropin-releasing factor (CRF) acts within the locus coeruleus (LC), to modulate activity of the LC-norepinephrine (NE) system. Combining molecular and cellular approaches, we demonstrate CRF receptor (CRFr) mRNA expression in Sprague-Dawley rat LC and provide the first in vivo evidence for agonist-induced internalization of CRFr. CRFr mRNA was detected in LC micropunches by RT-PCR. In dual labelling immunofluorescence studies, tyrosine hydroxylase (TH) containing neurons exhibited CRFr labelling. At the ultrastructural level, immunogold-silver labelling for CRFr was localized to the plasma membrane of TH-immunoperoxidase labelled dendrites. CRF (100 ng) injection into the LC produced a robust neuronal activation that peaked 10-15 min after injection and was maintained for the duration of the recording. This was associated with CRFr internalization in LC neurons that was apparent at 5 and 30 min after injection. By 5 min after injection the ratio of cytoplasmic to total dendritic CRFr-labelling was 0.81 +/- 0.01 in rats injected with CRF and 0.59 +/- 0.02 in rats injected with artificial cerebrospinal fluid (ACSF; P < 0.0001). Enhanced internalization of CRFr was maintained at 30 min after CRF injection, with the ratio being 0.86 +/- 0.02 for CRF-injected cases and 0.57 +/- 0.03 for ACSF-injected cases (P < 0.0001). Internalized CRFr was associated with early endosomes, indicative of degradation or recycling. Agonist-induced CRFr internalization in LC neurons may underlie acute desensitization to CRF or stress. This process may be a pivotal target by which stressors or pharmacological agents regulate the sensitivity of the LC-NE system to CRF and subsequent stressors.