Dynorphin and stress-related peptides in rat locus coeruleus: contribution of amygdalar efferents

Abstract

The interaction between the stress axis and endogenous opioid systems has gained substantial attention, because it is increasingly recognized that stress alters individual sensitivity to opiates. One site at which opiates and stress substrates may interact to have global effects on behavior is within the locus coeruleus (LC). We have previously described interactions of several opioid peptides [e.g., proopiomelanocortin, enkephalin (ENK)] with the stress-related peptide corticotropin-releasing factor (CRF) in the LC. To examine further the interactions among dynorphin (DYN), ENK, and CRF in the LC, sections were processed for detection of DYN and CRF or DYN and ENK in rat brain. DYN- and CRF-containing axon terminals overlapped noradrenergic dendrites in this region. Dual immunoelectron microscopy showed coexistence of DYN and CRF; 35% of axon terminals containing DYN were also immunoreactive for CRF. In contrast, few axon terminals contained both DYN and ENK. A potential DYN/CRF afferent is the central nucleus of the amygdala (CeA). Dual in situ hybridization showed that, in CeA neurons, 31% of DYN mRNA-positive cells colocalized with CRF mRNA, whereas 53% of CRF mRNA-containing cells colocalized with DYN mRNA. Finally, to determine whether limbic DYN afferents target the LC, the CeA was electrolytically lesioned. Light-level densitometry of DYN labeling in the LC showed a significant decrease in immunoreactivity on the side of the lesion. Taken together, these data indicate that DYN- and CRF-labeled axon terminals, most likely arising from amygdalar sources, are positioned dually to affect LC function, whereas DYN and ENK function in parallel.

Figure 1

Confocal immunofluorescence photomicrographs showing triple-labeling for DYN, CRF and TH in the coronal section through the LC. DYN immunoreactivity was labeled with rhodamine isothiocyanate (pseudocolored in magenta), CRF was labeled with fluorescein isothiocyanate (green) and TH was labeled with aminomethylcoumarin acetate (blue). A–C. High magnification photomicrographs of D showing DYN and CRF varicose processes overlapping TH neurons in the core LC (B–C) or peri-LC (A). D. Low magnification photomicrograph of A–C. Arrows indicate dorsal (D) and lateral (L) orientation of the tissue section. IV: fourth ventricle; scp: superior cerebellar peduncle. E–F. High magnification immunofluorescence images in a more rostral section through the LC showing processes containing DYN and CRF in close proximity to TH labeled perikarya. Straight arrows indicate processes containing both DYN and CRF (yellow). Scale bar, 250 μm.

Figure 2

Ultrastructural evidence for co-localization of DYN and CRF in the LC. A. An immunoperoxidase labeled axon terminal contains diffuse immunoreactivity for DYN, some of which is associated with dense core vesicles (dcv) and immunogold-silver labeling (arrowheads) for CRF. This terminal is surrounded by an astrocytic process (asterisks) and forms a symmetric synapse with an unlabeled dendrite (ud). B. A dually labeled CRF-DYN-axon terminal (CRF-DYN-t) forms an asymmetric synapse (black arrow) with an unlabeled dendrite (ud). Myelinated axons (ma) and unlabeled terminals (ut) can be seen in the neuropil. Scale bars, 0.5 μm.

Figure 3

DYN and CRF co-localized in amygdalar neurons innervate LC neurons. A. Brightfield micrograph showing labeling for DYN in the coronal section through the CeA. DYN immunolabeling can be identified in neurons and processes. Small arrows indicate individual DYN-labeled perikarya and processes. Arrows point dorsally (D) and laterally (L). B–C. Fluorescence photomicrographs illustrating CRF mRNA (green, B) and DYN mRNA (magenta, C) in the CeA. Arrows point dorsally (D) and laterally (L). D. A merged image of B and C showing coexistence of CRF mRNA and DYN mRNA (some examples are pointed by arrowheads) in the CeA. Scale bars, 100 μm.

Figure 4

Electrolytic lesions of the CeA yield decreases in DYN immunoreactivity in the LC. A. Schematic representation of three cases showing the boundaries of electrolytic lesions that were centered in the CeA. The extent of the individual lesions can be identified by ovals containing either light or dark gray shading or diagonal lines. An inset shows a schematic diagram adapted from the rat brain atlas of Swanson (1992) showing the level where the CeA was lesioned. Arrows indicate dorsal (D) and medial (M) orientation of the section illustrated. CeA, central nucleus of the amygdala B. Light level densitometry measurements of coronal sections through the LC taken from rats with unilateral electrolytic lesions of the CeA. Sections were processed for immunoperoxidase localization of DYN. Following an electrolytic lesion of the CeA, a significant decrease in the average density of DYN immunoreactivity was observed on the ipsilateral side of the lesion compared to the contralateral side (n=5). ^*, P < 0.05 vs contralateral side. C. DYN immunoreactivity (arrows) is shown on the ipsilateral side to the lesion. A significant decrease in DYN immunolabeling in the LC on the ipsilateral side to the lesion is noted. D. DYN immunoreactivity (arrows) is shown on the side contralateral to the lesion. scp, superior cerebellar peduncle. Scale bar Scale bars = 100 μm.

Figure 5

Confocal fluorescence photomicrographs showing DYN and ENK immunoreactivities in the coronal section through the peri LC (A–C) and LC core (D–F). A and D. DYN immunolabeling was detected by rhodamine isothiocyanate (pseudocolored in magenta). Arrows point to individual DYN-varicose processes that contain DYN. B and E. CRF was detected by fluorescein isothiocyanate (green). Arrows point to individual CRF-varicose processes that contain CRF. C and F. Merged images. Arrows point to DYN- and CRF-dual labeled varicose processes. Insets in panels C and F are high magnification images of the boxed regions. Scale bar, 100 μm.

Figure 6

Electron photomicrographs showing immunoperoxidase and immunogold-silver labeling for DYN and ENK, respectively, in the LC. A. A DYN-labeled axon terminal (DYN-t) enriched with mitochondria (m) is contacting (curved arrow) an unlabeled dendrite (ud). In the same field, an ENK-labeled axon terminal (ENK-t) forms a symmetric synapse (curved arrow) with an unlabeled dendrite (ud). B. A DYN-t and ENK-t are shown in the same field. C. A DYN-t contains dense core vesicles (dcv) and an ENK-t converge in a common unlabeled dendrite (ud). Also shown is an asymmetric synapse (curved arrow) and symmetric synapse (arrow) formed by DYN-t and ENK-t in a common dendrite (ud). D. An axon terminal exhibit both DYN and ENK (DYN+ENK-t) immunolabeling containing dense core vesicles (dcv). m, mitochodria; ma, myelinated axon. Scale bar, 0.5 μm.