Differential distribution patterns from medial prefrontal cortex and dorsal raphe to the locus coeruleus in rats

Abstract

Locus coeruleus (LC) consists of a densely packed nuclear core and a surrounding plexus of dendritic zone, which is further divided into several subregions. Whereas many limbic-related structures topographically target specific subregions of the LC, the precise projections from two limbic areas, that is, medial prefrontal cortex (mPFC) and dorsal raphe (DR), have not been investigated. The goal of the present study is to identify and compare the distribution patterns of mPFC and DR afferent terminals to the LC nuclear core as opposed to specific pericoerulear dendritic regions (Peri-LC). To address these issues, anterograde tracer injections were combined with dopamine-β-hydroxylase (DBH) immunofluorescent staining to reveal the distribution patterns around the LC nuclear complex. Our data suggest that both mPFC-LC and DR-LC projections exhibit selective afferent terminal patterns. More specifically, mPFC-LC projecting fibers mainly target the rostromedial Peri-LC, whereas DR-LC projecting fibers demonstrate a preference to the caudal juxtaependymal Peri-LC. Thus, our present findings provide further evidences that afferents to the LC are topographically organized. Understanding the relationship among different inputs to the LC may help to elucidate the organizing principle which likely governs the interactions between the broad afferent sources of the LC and its global efferent targets.

Figure 1

Color photomicrographs illustrate DBH or TH immunoreactive profiles in the LC (A-E; Cy3: red). It is quite obvious that DBH and TH immunoreactivity in the adjacent sections revealing highly similar labeling patterns (B-E). Extranuclear LC processes are mainly noted in the rostromedial and caudal juxtaependymal Peri-LC. These two subregions are marked in B-E (arrows) and also are clearly visualized in the horizontal section (A). DBH: dopamine-beta-hydroxylase; TH: tyrosine hydroxylase; LC: locus coeruleus; IV: the IVth ventricle. Scale bar: 80 μm.

Figure 2

Line drawing from reconstruction of anterograde tracer fluoro-ruby (FR) injection site in the mPFC is shown in A. A representative of FR (red) injection site is shown in B. Note that the spread of the tracer is restricted within the mPFC. PrL: prelimbic cortex; IL: infralimbic cortex; fmi: forceps minor of corpus callosum; mPFC: medial prefrontal cortex.

Figure 3

Color photomicrographs demonstrate the spatial relationship between mPFC-LC projecting fibers and DBH labeled LC (circled by dashed lines) in coronal sections. The left column shows DBH labeled LC profiles from rostral to caudal (A1-A3; Cy2: green). The middle column exhibits FR anterogradely labeled mPFC-LC projecting fibers and also a few FR retrogradely LC-mPFC projection neurons (B1-B3; red). The right column shows the merged photos. Note that the majority of mPFC-LC projecting fibers are located in the rostral sector of the LC, especially the rostromedial Peri-LC (marked by arrows in upper row), and very few fibers can be identified in the intermediate and caudal levels. In addition, a few FR labeled LC-mPFC projection neurons are noticed throughout all LC levels (marked by arrow heads). Scale bar: 30 μm.

Figure 4

Schematic line drawings illustrate the spatial relationship between mPFC-LC projecting fibers and LC nuclear core vs Peri-LC dendritic zones. Note that mPFC-LC descending fibers are mainly situated in the rostral sector of the LC, especially the rostromedial Peri-LC. LC: locus coeruleus; IV: 4^th ventricle.

Figure 5

Color photomicrographs demonstrate the spatial relationship between serotonin transporter (SERT) immunoreactive fibers and DBH positive LC nucleus (circled by dashed lines). The left column shows the LC from rostral to caudal (A1-A3) immunostained with DBH antiserum (Cy2: green). The middle column reveals SERT positive fibers from the same section (A2-C2; Cy3: red). The right column shows the merged photos. Note that SERT positive fibers are rather prevalent in the caudal juxtaependymal Peri-LC (marked by arrows in lower row). Scale bar: 60 μm.

Figure 6

Line drawing for the reconstruction of anterograde tracer injection site in the DR is shown in A. The fluorescent stained BDA (Cy2: green) reaction reveals the injection site in the DR (B). Note that a majority of BDA labeled cells are restricted in the DR. IV: 4th ventricle; LW: lateral wing; DM: dorsomedial; VM: ventromedial.

Figure 7

Color photomicrographs demonstrate the spatial relationship between DR-LC projecting fibers and DBH immunoreactive LC nucleus (circled by dashed lines). The left column shows LC profiles from rostral to caudal to reveal DBH immunostaining pattern (A1-A3; Cy2: green). The middle column reveals BDA anterogradely labeled DR-LC projecting fibers in the LC (B1-B3; Cy3: red). The right column shows the merged photos. The arrows point to the caudal juxtaependymal Peri-LC which contains the highest density of DR-LC projecting fibers. Scale bar: 40 μm.

Figure 8

Schematic line drawings illustrate the spatial relationship between DR-LC projecting fibers and LC nuclear core vs Peri-LC. Note that anterogradely labeled DR-LC projecting fibers exhibit a preference to the caudal juxtaependymal Peri-LC. LC: locus coeruleus; IV: 4^th ventricle.

Figure 9

Schematic diagram summarizes the overall topography of known afferent inputs to the LC. The area enclosed within the thin line denotes the LC nuclear core in the horizontal plane. Note that rostral Peri-LC tends to receive innervation from medial prefrontal cortex (mPFC), bed nucleus of stria terminals (BNST), central nucleus of amygadala (CNA), nucleus of solitary tract (NTS) and ventrolateral periaqueductal area (PAG). While mPFC and PAG mainly target the rostromedial Peri-LC, and CNA mainly targets the rostrolateral Peri-LC as well as BNST and NTS project to both rostromedial and rostrolateral Peri-LC. In contrast, DR is the only known structure to exhibit a preference to the caudal juxtaependymal Peri-LC. Additionally, nucleus paragigantocellularis (PGi), nucleus prepositus hypoglossi (PrH), Barrington's nucleus and the paraventricular nucleus of the hypothalamus appear to preferentially target the LC nuclear core. The precise distribution of afferents arising from other regions such as ventral tegmental area (VTA), median raphe (MR) and spinal cord etc. has yet to be determined. IV: 4^th ventricle. (the drawing is modified from Simpson and Lin, 2007).