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Descending projections from the rostral ventromedial medulla (RVM) to trigeminal and spinal dorsal horns are morphologically and neurochemically distinct - PubMed

Descending projections from the rostral ventromedial medulla (RVM) to trigeminal and spinal dorsal horns are morphologically and neurochemically distinct

Sue A Aicher et al. J Chem Neuroanat. 2012 Mar.

Abstract

Neurons in the rostral ventromedial medulla (RVM) are thought to modulate nociceptive transmission via projections to spinal and trigeminal dorsal horns. The cellular substrate for this descending modulation has been studied with regard to projections to spinal dorsal horn, but studies of the projections to trigeminal dorsal horn have been less complete. In this study, we combined anterograde tracing from RVM with immunocytochemical detection of the GABAergic synthetic enzyme, GAD67, to determine if the RVM sends inhibitory projections to trigeminal dorsal horn. We also examined the neuronal targets of this projection using immunocytochemical detection of NeuN. Finally, we used electron microscopy to verify cellular targets. We compared projections to both trigeminal and spinal dorsal horns. We found that RVM projections to both trigeminal and spinal dorsal horn were directed to postsynaptic profiles in the dorsal horn, including somata and dendrites, and not to primary afferent terminals. We found that RVM projections to spinal dorsal horn were more likely to contact neuronal somata and were more likely to contain GAD67 than projections from RVM to trigeminal dorsal horn. These findings suggest that RVM neurons send predominantly GABAergic projections to spinal dorsal horn and provide direct input to postsynaptic neurons such as interneurons or ascending projection neurons. The RVM projection to trigeminal dorsal horn is more heavily targeted to dendrites and is only modestly GABAergic in nature. These anatomical features may underlie differences between trigeminal and spinal dorsal horns with regard to the degree of inhibition or facilitation evoked by RVM stimulation.

Copyright © 2011 Elsevier B.V. All rights reserved.

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Conflict of interest statement

Conflict of interest statement: There are no conflicts of interest.

Figures

Figure 1
Figure 1

BDA injections into RVM produce anterograde labeling in trigeminal and spinal dorsal horns. (A) A representative micrograph of a BDA injection site in RVM and (B) a composite schematic illustrating individual BDA injections into RVM. (B) Each injection site is indicated by a grey shaded area, thus the darker regions represent areas included in multiple injections. All injections included the ventromedial medulla and all BDA injections into RVM resulted in bilateral labeling. The injection site outlined in red (B) represents the BDA injection site in the representative micrograph (A). (C) BDA labeling is present in representative micrographs in trigeminal (C) and spinal (C) dorsal horns. (C) BDA-labeled reticulomedullary projections were observed throughout the rostrocaudal extent of the trigeminal dorsal horn from Vi/Vc to Vc/C1 in laminae I and II as fibers with distinct varicosities (Arrows). (D) BDA-labeled reticulospinal projections were also observed as fibers and varicosities (Arrows) in laminae I and II of the lumbar spinal cord. The representative diagrams of the RVM (B), the trigeminal brainstem (C) and the spinal cord (D) are modified from the digital rat atlas of Paxinos and Watson (Paxinos and Watson, 1998) and are reproduced here with permission from the publisher. The dark rectangles in panels C and D represent the respective locations of each micrograph. The numbers on the upper right side of panels A, B and C represent the distance from bregma. In panel D, L5 refers to lumbar level 5 of the spinal cord. Scale bars: panels A and B = 1 mm; panels C and D = 100 μm. spV, spinal trigeminal tract.

Figure 2
Figure 2

Confocal micrographs show BDA-labeled reticulomedullary varicosities and GAD67 immunoreactivity in the trigeminal dorsal horn. (A) Reticulomedullary projections display axonal labeling with distinct varicosities. (B) GAD67 immunoreactivity in the trigeminal dorsal horn is abundant and predominantly punctate. (C) Overlay of panels A and B illustrate several BDA-labeled reticulospinal varicosities that contain GAD67 immunoreactivity (Yellow arrows). The representative diagram of the trigeminal brainstem in panel A is modified from the digital rat atlas of Paxinos and Watson (Paxinos and Watson, 1998) and is reproduced here with permission from the publisher. The white rectangle on the diagram in panel A represents the location of the micrographs. The number in the upper right of panel A represents the distance from bregma. The white arrows in the lower right of panel C represent the medial and dorsal orientation of the micrographs. Micrographs are 14 consecutive and overlapping optical sections composing a 6.5 um thick Z stack. Scale bar = 10 um.

Figure 3
Figure 3

Reticulomedullary and reticulospinal terminals were observed contacting neurons with confocal (A-C) and electron (D) microscopy. (A-C) Confocal micrographs (10 consecutive and overlapping optical sections composing a 4.5 um thick Z stack) illustrate (A) a NeuN-ir neuron and (B) BDA-labeled reticulomedullary varicosities in the trigeminal dorsal horn. (C) The overlay demonstrates appositions between two BDA-labeled reticulomedullary varicosities and the NeuN-ir cell. Scale bar = 10 um for panels A - C. (D) Electron micrograph illustrating a BDA-labeled terminal (BDA-t) forming an apposition with an unlabeled perikarya (up). An unlabeled terminal (ut) is also seen forming an apposition with the cell. Scale bar = 500 nm.

Figure 4
Figure 4

Electron micrographs illustrate the frequently observed types of contacts seen between GABAergic reticulomedullary (A, B) and reticulospinal terminals (C) onto unlabeled dendrites. (A) A GAD67-ir BDA-labeled reticulomedullary terminal (BDA+GAD-t) contacts a large unlabeled dendrite (ud). The dendrite also receives asymmetric synapses (curved arrows) from two unlabeled axon terminals (ut). (B) A small unlabeled dendrite (ud) receives an apposition from a GAD67-ir reticulomedullary terminal (BDA+GAD-t) in the trigeminal dorsal horn. (C) Also the most common interaction seen in the lumbar dorsal horn, a GAD67-ir BDA-labeled terminal (BDA+GAD-t) forms a symmetric synapse (curved arrow) with an unlabeled dendrite (ud). All scale bars = 250 nm.

Figure 5
Figure 5

Electron micrographs illustrate less common occurrences of BDA and GAD67 immunoreactivity in the trigeminal (A-C) and lumbar (D) dorsal horns. (A) A GAD67-ir BDA-labeled reticulomedullary terminal (BDA+GAD-t) contacts a GAD67-ir dendrite (GAD-d). A BDA-labeled axon (BDA-a), a GAD67-ir axon (GAD-a), and an unlabeled dendrite (ud) are also seen in the image. (B) A BDA-labeled reticulomedullary terminal (BDA-t) is largely surrounded by a astrocytic glial processes (asterisks). (C) GAD67-ir axons (GAD-a) and an unlabeled terminal (ut). (D) Two GAD67-ir BDA-labeled reticulospinal terminals (BDA+GAD-t) are seen in proximity to a GAD67-ir cell (GAD-p). The terminal at the bottom left of the image is in contact with the cell while the terminal in the upper right is separated by a glial sheath (asterisk). Scale bars for panels A-C = 250 nm, and the scale bar for panel D = 1 um.

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