Amygdalar peptidergic circuits regulating noradrenergic locus coeruleus neurons: linking limbic and arousal centers

Abstract

The endogenous opioid peptides, met- or leu-enkephalin, and corticotropin-releasing factor (CRF) regulate noradrenergic neurons in the locus coeruleus (LC) in a convergent manner via projections from distinct brain areas. In contrast, the opioid peptide dynorphin (DYN) has been shown to serve as a co-transmitter with CRF in afferents to the LC. To further define anatomical substrates targeting noradrenergic neurons by DYN afferents originating from limbic sources, anterograde tract-tracing of biotinylated dextran amine (BDA) from the central amygdaloid complex was combined with immunocytochemical detection of DYN and tyrosine hydroxylase (TH) in the same section of tissue. Triple labeling immunocytochemistry was combined with electron microscopy in the LC where BDA was identified using an immunoperoxidase marker, and DYN and TH were distinguished by the use of sequential immunogold labeling and silver enhancement to produce different sized gold particles. Results show direct evidence of a monosynaptic pathway linking amygdalar DYN afferents with LC neurons. To determine whether DYN-containing amygdalar LC-projecting neurons colocalize CRF, retrograde tract-tracing using fluorescent latex microspheres injected into the LC was combined with immunocytochemical detection of DYN and CRF in single sections in the central amygdala. Retrogradely labeled neurons from the LC were distributed throughout the rostro-caudal extent of the central nucleus of the amygdala (CeA) as previously described. Cell counts showed that approximately 42% of LC-projecting neurons in the CeA contained both DYN and CRF. Taken with our previous studies showing monosynaptic projections from amygdalar CRF neurons to noradrenergic LC cells, the present study extends this by showing that DYN and CRF are co-transmitters in monosynaptic projections to the LC and are poised to coordinately impact LC neuronal activity.

Figure 1

A. Low magnification brightfield photomicrograph of a representative BDA injection into the CeA in the rat brain. Arrows indicate neurons exhibiting peroxidase labeling for BDA at the injection site. B. A schematic diagram adapted from the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986) showing the anterior posterior level of the region of the central amygdaloid complex targeted for injection placement. Arrows indicate dorsal (D) and medial (M) orientation. Panel C shows a higher magnification view of panel A and reflects the level shown in the schematic from panel B. D. Electron photomicrograph showing immunoperoxidase labeling for biotinylated dextran amine (BDA) and dual immunogold-silver labeling for dynorphin (DYN; large gold) and tyrosine hydroxylase (TH; small gold) in the locus coeruleus. Dense peroxidase labeling can be seen in a BDA-labeled axon terminal containing large gold-silver grains (arrowheads) that indicate labeling for DYN (BDA+DYN-t). In a separate profile, a dendrite containing TH.can be seen by the presence of small gold-silver labeling. E. A BDA+DYN-t forms a symmetric synapse (curved arrow) with a dendrite that lacks detectable immunoreactivity for TH. F. Two DYN-labeled axon terminals that do not contain BDA can be seen in proximity to a BDA+DYN-t. G. A BDA-labeled terminal and a BDA+DYN-t converge on a TH-labeled dendrite. A BDA-labeled terminal forms a symmetric synapse (curved arrow) while BDA+DYN-t forms an asymmetric synapse (arrows) with a dendrite. ud, unlabeled dendrite. CeA, central nucleus of the amygdala; int, internal capsule; son, supraoptic nucleus. Scale bar for panel C = 100 μm. Scale bar for panels D–G = 0.50 μm

Figure 2

Electron photomicrograph showing immunoperoxidase labeling for biotinylated dextran amine (BDA) and dual immunogold-silver labeling for dynorphin (DYN) and tyrosine hydroxylase (TH) in the locus coeruleus. A–B. Two adjacent sections showing a BDA-labeled axon terminal containing dynorphin (BDA+DYN-t) contacting a TH-labeled dendrite (TH-d). DYN-t is labeled with large gold particles (arrowheads) while TH-t is labeled with small gold particles (arrows). C. A BDA+DYN-t and a BDA-labeled axon terminal (BDA-t) converge on a TH-d. The BDA-t is also apposed to an unlabeled terminal (ut) that also contacts the TH-d. D. A BDA+DYN-t is seen contacting a TH-d, and is also apposed to an unlabeled axon terminal (ut). E. A BDA+DYN-t forms a symmetric synapse (curved arrow) with a TH-d. F. A BDA+DYN-t forms an asymmetric synapse (multiple arrows) with a TH-d. Arrows point to small immunogold-silver particles that indicate TH immunoreactivity. Arrowheads point to large immunogold silver particles that denote DYN immunoreactivity. Scale bars = 0.05 μm.

Figure 3

A. Photomicrograph showing a representative tracer injection of green fluorescent latex microspheres into the LC. B. Photomicrograph showing representative retrogradely labeled neurons in the CeA following an injection into the LC. Insets in panels A and B show schematic diagrams adapted from the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986) indicating the anterior posterior level of the representative injection site in the LC and retrograde labeling in the CeA. Arrows indicate dorsal (D) and medial (M) orientation of the sections. Arrowheads point to retrogradely labeled neurons in the CeA. CeA, central nucleus of the amygdala; LC, locus coeruleus; 4V, fourth ventricle; scp, superior cerebellar peduncles. Scale bar, 100 μm.

Figure 4

High magnification photomicrographs illustrating retrograde transport from the LC to the CeA. Retrogradely labeled neurons in the CeA (A and E) contain DYN (B and F) and CRF (C and G). DYN immunolabeling was detected using a rhodamine isothiocyanate-tagged secondary antibody (red; B and F). Arrowheads point to individual DYN-immunoreactive neurons that contain only DYN. CRF immunolabeling was detected using a Cy5-tagged secondary antibody (blue; C and G). Arrowheads point to individual CRF-immunoreactive neurons that contain only CRF. D and H are merged images of A–C and E–G, respectively. Arrows point to single neurons that contain CRF, DYN and retrobeads. Arrowheads denote neurons that exhibit retrobeads, DYN or CRF only. Scale bar, 100 μm.