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

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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 firstin vivoevidence 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.