Agonist-induced internalization of corticotropin-releasing factor receptors in noradrenergic neurons of the rat locus coeruleus.
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.
Reyes
Beverly A S
BA
Department of Neurosurgery, Farber Institute for Neurosciences, Thomas Jefferson University, 900 Walnut Street, Suite 400, Philadelphia, PA 19107, USA. bsr103@jefferson.edu
Fox
Krysta
K
Valentino
Rita J
RJ
Van Bockstaele
Elisabeth J
EJ
D000818:Animals; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D007150:Immunohistochemistry / Q000379:methods; D008125:Locus Coeruleus / Q000166:cytology*; D008297:Male; D016253:Microscopy, Immunoelectron / Q000379:methods; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D009638:Norepinephrine / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018019:Receptors, Corticotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D012333:RNA, Messenger; D018019:Receptors, Corticotropin-Releasing Hormone; D003346:Corticotropin-Releasing Hormone; D014446:Tyrosine 3-Monooxygenase; D009638:Norepinephrine