Spinal projections of the A5, A6 (locus coeruleus), and A7 noradrenergic cell groups in rats

Abstract

The pontine noradrenergic cell groups, A5, A6 (locus coeruleus), and A7, provide the only noradrenergic innervation of the spinal cord, but the individual contribution of each of these populations to the regional innervation of the spinal cord remains controversial. We used an adeno-associated viral (AAV) vector encoding green fluorescent protein under an artificial dopamine beta-hydroxylase (PRSx8) promoter to trace the spinal projections from the A5, A6, and A7 groups. Projections from all three groups travel through the spinal cord in both the lateral and ventral funiculi and in the dorsal surface of the dorsal horn, but A6 axons take predominantly the dorsal and ventral routes, whereas A5 axons take mainly a lateral and A7 axons a ventral route. The A6 group provides the densest innervation at all levels, and includes all parts of the spinal gray matter, but it is particularly dense in the dorsal horn. The A7 group provides the next most dense innervation, again including all parts of the spinal cord, but is it denser in the ventral horn. The A5 group supplies only sparse innervation to the dorsal and ventral horns and to the cervical and lumbosacral levels, but provides the densest innervation to the thoracic intermediolateral cell column, and in particular to the sympathetic preganglionic neurons. Thus, the pontine noradrenergic cell groups project in a roughly topographic and complementary fashion onto the spinal cord. The pattern of spinal projections observed suggests that the locus coeruleus might have the greatest effect on somatosensory transmission, the A7 group on motor function, and the A5 group on sympathetic function.

Figure 1

Double labeling of injection sites for dopamine β-hdrosylase (DBH) immunoreactivity (red; A,D,G) and green fluorescent protein (GFP)-labeling (green; B,E,H) and merged images (showing doubly labeled neurons in yellow) after injections into the LC (A–C), A7 (D–F) and A5 (G–I) cell groups. While most of the GFP-labeled neurons were also immunoreactive for DBH (closed arrows), a few small GFP-labeled cells were seen (open arrows) that were not DBH-immunoreactive. 4V, fourth ventricle; Bar, Barrington’s nucleus; SubCA, subcoeruleus area. Scale bars: 0.1 mm. A magenta-green copy of this figure is available as Supplementary Figure 1.

Figure 2

A series of immunofluorescence photomicrographs showing that GFP-labeled axons in the spinal cord were also DBH-immunoreactive. GFP-immunoreactive axons are shown in green (A, D,G,J,M), DBH-labeled axons in red (B,E,H,K,N), and overlap in yellow (C,F,I,L,O). Images show staining of axons from LC running through the dorsal horn (A–C), and ventral funiculus (D–F), from A7 in the ventral horn (G–I; J–L), and from A5 in the intermediolateral cell column (IML) (M–O). Note that all of the GFP stained axons also contained DBH immunoreactivity. However, the GFP staining fills the axons, whereas that DBH immunoreactivity in thin ramifying axons is associated with mainly with varicosities. Scale bar for all images: 0.02 mm. A magenta-green copy of this figure is available as Supplementary Figure 2.

Figure 3

A series of drawings to illustrate the distribution of labeled neurons in the injection sites in the A7 (left), LC (center) and A5 (right) groups in three representative animals. Every dot represents a single GFP-ir cell stained with the immunoperoxidase method in one representative case for each injection site. DR, dorsal raphe nucleus; ICol, inferior colliculus; KF, Kölliker-Fuse nucleus;MoV, motor trigeminal nucleus; PB, parabrachial nucleus; PPT, pedunculopontine tegmental nucleus; PSV, principal sensory trigeminal nucleus; SNV, spinal trigeminal nucleus; SO, superior olivary nucleus; VIIn, facial nerve

Figure 4

Drawings of labeled axons in the spinal cord of one representative case from A7 (left), LC (center) and A5 (right) injections, respectively. The spinal cord is shown at Cervical (C7-8), Upper thoracic (T1-2), Lower thoracic (T8-9) and Lumbar (L3-4) levels. For the LC and A7 cases, axons observed in one single section are shown. Because of the sparse spinal innervation found in A5 injection cases, axons observed in 3 consecutive sections were superimposed into one illustration.

Figure 5

A series of high magnification photomicrographs showing GFP-immunoreactive axons descending through horizontal sections of the spinal cord. (A) After an injection into the LC descending axons are seen running longitudinally in the superficial layers of the dorsal horn at the cervical level. (B) After an A7 injection, a dense meshwork of labeled fibers is seen in the ventral horn at the cervical level. (C) After an A5 injection, dense innervation is labeled in the thoracic IML. Scale bars: 0.1 mm.

Figure 6

A series of high magnification photomicrographs of transverse spinal sections showing GFP-labeled axons. (A) After an injection into the LC, the density of the descending axons is seen running through the superficial layers of the cervical (C7-8) dorsal horn. (B) After an injection in the A7 cell group, dense projections are seen in the cervical (C7-8) spinal cord in the lamina IX of the ventral horn, ramifying among the motor neurons. (C) After an injection into the A5 cell group, there is dense innervation of the IML at the upper thoracic level (T1-2). Scale bars: 0.05 mm.

Figure 7

Photomicrographs showing the relationship of the GFP-ir fibers (black) to cholinergic neurons (brown staining for ChAT) in the spinal cord. LC axons in the dorsal horn were observed along its superficial margin with the Lissauer’s tract (LT) (panel A), and were numerous throughout the dorsal horn, especially at its base adjacent to the dorsal columns (DC) (panel B). In the ventral horn, labeled LC axons were not located adjacent to cholinergic neurons (C). In contrast, anterogradely labeled axons from A7 neurons ran along the surface of the cell bodies and proximal dendrites of ventral horn cholinergic neurons, as shown in two series of photomicrographs taken at 3 different focal planes through a pair of cholinergic neurons (D–F and G–I), respectively. Arrows point to black anterogradely labeled axons and terminals. Following A5 injection, labeled axons showed intense ramification and numerous boutons along the cell bodies and proximal dendrites of cholinergic neurons in the IML at the thoracic level (J and K are two focal planes through the same neurons, L is a different field). Scale bar: 50 μm for A–C; 25 μm for D–L.

Figure 8

A series of confocal z-stack projections of sections through the spinal cord stained red for choline acetyltransferease (ChAT) and green for GFP. A, D, and G show sections after an A5 injection; B, E, H after an LC (A6) injection; and C, F, I after an A7 injection. The upper row (A–C) demonstrate the extent of innervation of the IML after injections in each cell group. There is much more extensive, fine branching axonal arborization in the IML after the A5 injection. On single 1 μm sections (see fig. 9), they form boutons that are in close proximity to the dendrites and, to a lesser extent, cell bodies of IML neurons. LC and A7 axons travel in proximity to the IML, and form occasional boutons, but they do not show as much arborization in the IML. The second row (D–F) shows the innervation of the ventral horn (VH) after these injections. Large amounts of fine axonal arborization and boutons in proximity to ventral horn neurons were seen only after A7 injections. G shows another section through the IML after an A5 injection, in which the innervation is clearly related to proximal dendrites rather than cell bodies. H shows LC axons in relation to two small cholinergic interneurons in the dorsal horn (DH). The LC axons are in close proximity, but do not arborize and have few boutons in proximity to these cells. I shows A7 axons arborizing and forming boutons among cremasteric motor neurons at the L1 level of the spinal cord. This cell group was innervated by both A5 and A7 axons, but neither innervated the cremasteric neurons as intensively as their primary targets. Scale bar in I = 20 μm. A magenta-green copy of this figure is available as Supplementary Figure 3.

Figure 9

A series of non-consecutive 1 μm thick confocal sections through the IML after an A5 injection (A–D) and through the ventral horn after an A7 injection (E–H). These were part of the same z-stacks shown in fig. 8G and F, respectively. The thin focal planes show that the individual noradrenergic axons (green) coursed along the surface of cholinergic cell bodies and dendrites (red), giving off numerous boutons (arrows). Scale bar = 20 μm. A magenta-green copy of this figure is available as Supplementary Figure 4.