Sex differences in the anatomical and functional organization of the periaqueductal gray-rostral ventromedial medullary pathway in the rat: a potential circuit mediating the sexually dimorphic actions of morphine

Abstract

Previous studies have demonstrated that morphine, administered systemically or directly into the periaqueductal gray (PAG), produces a significantly greater degree of antinociception in males in comparison with females. Because the midbrain PAG and its descending projections to the rostral ventromedial medulla (RVM) constitute an essential neural circuit for opioid-based analgesia, the present studies were conducted to determine whether sex differences in the anatomical organization of the PAG-RVM pathway, and its activation during persistent inflammatory pain, could account for sex-based differences in opioid analgesia. In the rat, retrograde tracing was combined with Fos immunocytochemistry to investigate sexual dimorphism in the organization of the PAG-RVM circuit and its activation by persistent inflammatory pain induced by intraplantar injection of complete Freund's adjuvant (CFA). The ability of morphine to suppress the activation of the PAG-RVM circuit was also examined. Sexually dimorphic retrograde labeling was observed within the dorsomedial and lateral/ventrolateral PAG at all rostrocaudal levels, with females having significantly more PAG-RVM output neurons in comparison with males. While no sex differences were noted in the activation of the PAG by persistent inflammatory pain, significantly more PAG-RVM cells were activated in males in comparison with females. Systemic administration of morphine significantly suppressed CFA-induced Fos in the PAG in males only. The results of these studies demonstrate that both the anatomical organization and the functional activation of the PAG-RVM circuit are sexually dimorphic and may provide the anatomical substrate for sex-based differences in morphine analgesia.

Figure1

Representative examples of FG injection into the RVM of a male (top) and female (bottom) rat. Scale bar: 200μm.

Figure 2

Distribution of cells retrogradely labeled from the rostral ventromedial medulla in males and females at six rostrocaudal levels of the periaqueductal gray. Each black circle represents one FG-immunoreactive cell. Bar graphs show the mean number (± S.E.M.) of FG-immunoreactive cells for the dorsomedial and lateral/ventrolateral regions of PAG.

Figure 3

Photomicrographs of cortical retrograde labeling in the male (A) and female (B) rat (Bregma –1.0). Scale bar: 200μm. Bar graph (C) shows the mean number (+ S.E.M.) of retrogradely labeled cells in the cortex of males and females. Labeling was localized to pyramidal cells in layer 5. Dorsal counts include primarily motor cortex, while ventral counts are primarily somatosensory cortex.

Figure 4

Distribution of CFA induced Fos within the periaqueductal gray of males and females across six rostrocaudal levels. Each black circle represents one Fos+ cell. No sex differences were noted in either the distribution or number of Fos+ cells for males and females.

Figure 5

Distribution of PAG-RVM output neurons that expressed CFA-induced Fos for males and females across six rostrocaudal levels of the PAG. Each black circle represents one double-labeled (FG+Fos) cell. Bar graphs display the mean number of FG/Fos cells (± S.E.M.) for each level and region.

Figure 6

Color photomicrograph showing a low (A) and high (B) power example of single and double labeled Fos and FG labeled cells in the PAG of a male rat. Scale bar = 100 μm in panel A; scale bar = 50 μm in panel B.

Figure 7

Percentage of PAG-RVM neurons that were activated by persistent inflammatory pain (FG+Fos) in males (left) and females (right) throughout the rostrocaudal axis of PAG.

Figure 8

Percentage of Fos+ PAG neurons that were retrogradely labeled from the RVM (Fos+FG) in males (left) and females (right) throughout the rostrocaudal axis of PAG.

Figure 9

Distribution of CFA-induced Fos (black dots) following saline (black) or morphine (white) administration in males (left) and females (right) along the rostrocaudal axis of PAG. Each black circle represents one Fos+ cell. Bar graphs show mean number of Fos+ cells (± S.E.M.) for each treatment group broken down separately for the dorsomedial and lateral/ventrolateral PAG.

Figure 10

Percentage of PAG-RVM neurons expressing CFA-induced Fos (%FG/Fos) following saline (black bars) or morphine (4.5 mg/kg; s.c.; white bars) in male (left) and female (right) rats for six rostrocaudal levels of PAG.

Figure 11

Percentage of CFA-induced Fos positive neurons that were retrogradely labeled from the RVM (%Fos/FG) following saline (black bars) or morphine (4.5 mg/kg; s.c.; white bars) in male (left) and female (right) rats for six rostrocaudal levels of PAG.