Periaqueductal gray neurons project to spinally projecting GABAergic neurons in the rostral ventromedial medulla

Abstract

The analgesic effects of morphine are mediated, in part, by periaqueductal gray (PAG) neurons that project to the rostral ventromedial medulla (RVM). Although much of the neural circuitry within the RVM has been described, the relationship between RVM neurons and PAG input and spinal output is not known. The objective of this study was to determine whether GABAergic output neurons from the PAG target RVM reticulospinal neurons. Immunocytochemistry and confocal microscopy revealed that PAG neurons project extensively to RVM neurons projecting to the spinal cord, and two-thirds of these reticulospinal neurons appear to be GABAergic (contain GAD67 immunoreactivity). The majority (71%) of PAG fibers that contact RVM reticulospinal GAD67-immunoreactive neurons also contained GAD67 immunoreactivity. Thus, there is an inhibitory projection from PAG to inhibitory RVM reticulospinal neurons. However, there were also PAG projections to the RVM that did not contain GAD67 immunoreactivity. Additional experiments were conducted to determine whether the heterogeneity in this projection can be explained by the electrophysiological character of the RVM target neurons. PAG projections to electrophysiologically defined and juxtacellularly filled ON, OFF, and Neutral cells in the RVM were examined. Similar to the pattern reported above, both GAD67- and non-GAD67-immunoreactive PAG neurons project to RVM ON, OFF, and Neutral cells in the RVM. These inputs include a GAD67-immunoreactive projection to a GAD67-immunoreactive ON cell and non-GAD67 projections to GAD67-immunoreactive OFF cells. This pattern is consistent with PAG neurons producing antinociception by direct excitation of RVM OFF cells and inhibition of ON cells.

Figure 1

Micrographs of representative injection sites into cervical spinal cord (A) and ventrolateral PAG (B), as well as micrographs of representative labeling of FG (C) and PHA-L in RVM (D). (A) Epifluorescent micrograph of FG injection into cervical spinal cord. Injection is apparent in the left dorsal horn region. Arrow indicates substantia gelatinosa (SG). (B) Epifluorescent micrograph of PHA-L injection site in ventrolateral PAG (VLPAG). Arrow indicates injection in the left VLPAG. Micropunches placed in tissue to distinguish left and right sides are visible in the lower right portion of tissue sections (A,B). The PAG is outlined by the dashed line. Aq = aqueduct. (C,D) Epifluorescent micrographs of FG (C)and PHA-L (D) labeling in RVM. Arrows indicate PHA-L fibers (D), which are abundant throughout RVM. pyr = pyramidal tract. Scale bars for A = 1.0 mm; B = 2.0 mm; C,D = 500 μm. Directional arrows indicate dorsal (D) and medial (M) for all panels.

Figure 2

Anterogradely labeled PAG fibers contact retrogradely labeled reticulospinal neurons in RVM. (A) Micrograph illustrating the level and area of RVM used for analysis, as indicated by the box. pyr = pyramidal tract. (B) Epifluorescent micrograph illustrating the overlapping distribution of retrogradely labeled FG neurons (green) and anterogradely labeled PHA-L fibers (red). Arrow indicates a PHA-L fiber. (C) Approximately one-third of the reticulospinal neurons identified in this study received appositions from PAG varicose fibers labeled in the same case (see text for detailed analysis). Arrow indicates a PHA-L varicosity (red) apposed to a FG neuron (green). The image is confocal Z projection of ten 0.8 μm optical sections with 0.4 μm of overlap forming a 4.4 μm thick stack. Scale bar for A = 2.0 mm, B = 500 μm; C = 20 μm. Directional arrows indicate dorsal (D) and medial (M) for all panels.

Figure 3

GAD67 immunoreactivity is present in a subset of RVM reticulospinal neurons. RVM neurons are retrogradely labeled from the spinal cord with FG (A). (B) Immunostaining for GAD67 (red) in RVM. (C) An overlay of both markers shows clear colocalization between reticulospinal neurons (FG) and GAD67 immunoreactivity, as well as neurons that did not contain GAD67 immunoreactivity. The arrow indicates a reticulospinal neuron (A), GAD67 immunoreactivity of the same neuron (B) and the colocalization of both markers in the overlay (C). The image is a single confocal section, 0.8 mm thick. Scale bar = 20 μm.

Figure 4

PAG fibers often target neurons in RVM that contain GAD67 immunoreactivity. (A) PHA-L varicose fibers (red) appose retrogradely labeled FG reticulospinal neurons (green), as indicated by the two arrows. (B, C) GAD67 immunoreactivity (purple) is apparent in the region and both FG neurons that receive PAG input are also GAD67 immunoreactive. The arrowhead in all three panels indicates a FG-labeled reticulospinal neuron that does not contain GAD67 immunoreactivity nor receives input from PAG. Of the 72 FG cells that received PAG input, 63% (45/72) of the target neurons contained GAD67 immunoreactivity. Images are a confocal Z projection of twelve 0.8 μm optical sections with 0.4 μm of overlap forming a 5.2 μm thick stack. Scale bar = 20 μm.

Figure 5

PAG fibers containing GAD67 immunoreactivity target GAD67 immunoreactive reticulospinal neurons in RVM. (A) A PHA-L varicose fiber (red) is apposed to a reticulospinal FG neuron (green; arrow). (B) GAD67 immunoreactivity (purple) is seen in the same area. (C) GAD67 immunoreactivity is colocalized within both the reticulospinal neuron and the PHA-L varicosity (arrow), is apparent in the overlay. Of the 45 FG and GAD67 immunoreactive cells that received PHA-L input, 32 (71%) received PAG input colocalized with GAD67 immunoreactivity. In contrast, of the 27 FG-only cells that received PAG input, only 13 (48%) received PAG input colocalized with GAD67 immunoreactivity. Image is a confocal Z projection of eight 0.8 μm optical sections with 0.4 μm of overlap forming a 3.6 μm thick stack. Scale bar = 20 μm.

Figure 6

Most PAG fibers apposed to a Neutral cell in the RVM do not contain GAD67 immunoreactivity. (A) A biotinamide-filled neuron in RVM, electrophysiologically characterized as a Neutral cell. The following three panels are magnified images of the boxed area. Scale bar = 50 μm. (B, C, D) The arrowhead in the three panels indicates a PHA-L varicose fiber (green) apposed to the Neutral biotinamide-filled cell (red) and, while GAD67 immunoreactivity (purple) is present in the region, there is no colocalization between the PHA-L varicosity and GAD67 immunoreactivity. Colocalization between the biotinamide-filled cell and GAD67 immunoreactivity is apparent in the overlay (D). The images are a confocal Z projection of ten 0.8 μm optical sections with 0.4 μm of overlap forming a 4.4 μm thick stack. Scale bar = 20 μm.

Figure 7

PAG fibers apposed to an ON cell in the RVM. Both the ON cell and the PAG fibers contain GAD67 immunoreactivity. (A) A biotinamide-filled neuron (red), functionally characterized as an ON cell, as well as PHA-L fibers (green; arrow). (B) GAD67 immunoreactivity is present in the same region (purple); the arrow points to the GAD67 immunoreactivity of the previously indicated PHA-L apposition (A). (C) Colocalization between GAD67 immunoreactivity and both the ON cell as well as the PHA-L apposition (arrow) is apparent in the overlay. Of the three functionally characterized ON cells, all of them contained GAD67 immunoreactivity and received PAG input. Images are a confocal Z projection of twenty 0.8 μm optical sections with 0.4 μm of overlap forming an 8.4 μm thick stack. Scale bar = 20 μm.

Figure 8

PAG fibers apposed to an OFF cell in the RVM. The OFF cell contains GAD67 immunoreactivity, but the PAG fibers do not. A PAG varicose fiber (green) apposes a biotinamide-filled OFF cell (arrowhead; A). GAD67 immunoreactivity (purple) is in the same region (B). GAD67 immunoreactivity is found in the OFF cell (C; inset D), but not in the PHA-L varicosity (arrowhead). Of the three functionally characterized OFF cells containing GAD67 immunoreactivity, all three received PAG input; however, none of the PAG inputs contained GAD67 immunoreactivity. Image is a confocal Z projection of twenty-four 0.8 μm optical sections with 0.4 μm of overlap forming a 10 μm thick stack. Scale bars for A, B, C = 50 μm; D = 15 μm.