Differential activation of the periaqueductal gray by mild anxiogenic stress at different stages of the estrous cycle in female rats

Abstract

The effect of acute exposure to mild anxiogenic stress on cutaneous nociceptive threshold was investigated in female Wistar rats at different stages of the estrous cycle. Baseline tail flick latencies did not change significantly during the cycle. However after brief exposure to vibration stress (4 Hz for 5 min), rats in late diestrus, but not at other cycle stages, developed a hyperalgesia (decrease in tail flick latency). Animals in late diestrus revealed a more than fivefold increase in the density of Fos-like immunoreactive nuclei in the dorsolateral, lateral, and ventrolateral columns in the caudal half of the periaqueductal gray matter (PAG). There was no change in the density of Fos-like immunoreactive nuclei in the PAG in rats in estrus and early diestrus, although rats in proestrus showed a smaller (50%) but significant increase. Rats undergoing withdrawal from a progesterone dosing regimen (5 mg/kg i.p. twice daily for 6 days) designed to mimic the fall in progesterone that occurs naturally during late diestrus, exhibited a stress-induced hyperalgesia that was similar to animals in late diestrus and a significant increase in Fos-positive cells in the PAG. We suggest that falling levels of progesterone during late diestrus may be a predisposing factor for the development of stress-induced hyperalgesia, which is linked to differential activation of descending pain control circuits in the PAG. Similar changes in women, when progesterone levels fall during the late luteal phase of the menstrual cycle, may contribute to the development of premenstrual symptoms that include increased anxiety and hyperalgesia.

Figure 1

(a–d) Characteristic cytological appearance of vaginal smears from spontaneously cycling rats. (a) Proestrus, (b) estrus, (c) early diestrus, (d) late diestrus, and (e) smear from a rat after 7 days of progesterone treatment (5 mg/kg i.p. twice daily). (f) Smear taken 24 h after withdrawal from progesterone.

Figure 2

Schematic representation of method used to quantify Fos-like immunoreactive profiles in the periaqueductal gray matter (PAG). (a) Low-power photomicrograph of a section through the caudal PAG. High-magnification inset shows individual stained nuclei. (b) Computer-generated plot of all immunostained nuclei recognized in this section. (c) Outline diagrams of sections at the four levels (I–IV) of the PAG sampled. To calculate the density of Fos-positive nuclei, we orientated triangular counting frames representing an area of 60 000 μm² in the tissue over different columns of the PAG on the computer-generated images of representative sections from each rostrocaudal level. Numbers below sections indicate distance caudal to bregma. D, dorsal; DL, dorsolateral; LI, lateral (I); LII, lateral (II); and VL, ventrolateral.

Figure 3

Graph shows time course of the effect of 5 min of vibration stress (gray bar) on tail flick latency (TFL) in female rats at different stages of the estrous cycle. All values (mean±SEM) are expressed as a percentage of mean prestress baseline values (n=5–6 per group). Control groups were not exposed to vibration stress. ^*p<0.05, post hoc Dunnett's test in comparison to baseline readings after significant (p<0.05) repeated measures one-way ANOVA applied to raw data. F(8,4,32)=3.531, p=0.0048.

Figure 4

Mean density of Fos-like immunoreactive nuclei in the whole periaqueductal gray matter (PAG) of spontaneously cycling female rats in the stressed and control groups (n=5–6 per group) and in naive rats (n=3 per cycle stage), which had not undergone any behavioral testing procedures. Abbreviations as in Table 1. NB Because a priori estrous cycle stage determines responsiveness to stress (see Figure 3), we restricted analysis to within cycle stages. All values mean±SEM; ^*p<0.05. ^**p<0.01, ^***p<0.001, post hoc Bonferroni test after significant (p<0.05) one-way ANOVA within each cycle stage. Proestrus: F(2,10)=33.430, p<0.0001; late diestrus: F(2,10)=7.831, p=0.009.

Figure 5

Effect of exposure to 5 min vibration stress on the density of Fos-immunoreactive cells in individual columns of the periaqueductal gray matter (PAG) at rostral (levels I and II) and caudal (levels III and IV) levels in rats in late diestrus. Cartoon depicts areas of PAG sampled (for more details see Figure 2). Histograms show mean Fos immunoreactive cell density indifferent columns of the PAG. All values mean±SEM; ^*p<0.05, ^**p<0.01, post hoc Bonferroni test in comparison to the control group after significant (p<0.05) one-way ANOVA. DL: F(3,16)=4.772, p=0.015; LI: F(3,16)=5.137, p=0.011; LII: F(3,16)=5.030, p=0.012; VL: F(3,16)=4.561, p=0.017.

Figure 6

Effect of exposure to 5 min vibration stress on the density of Fos-immunoreactive cells in individual columns of the periaqueductal gray matter (PAG) at rostral (levels I and II) and caudal (levels III and IV) levels in rats in proestrus. Cartoon depicts areas of PAG sampled (for more details see Figure 2). Histograms show mean Fos immunoreactive cell density in different regions of the PAG. All values mean±SEM; ^*p<0.05, ^**p<0.01, post hoc Bonferroni test in comparison to the control group after significant (p<0.05) one-way ANOVA. VL: F(3,16)=8.454, p=0.001.

Figure 7

Graph shows time course of the effect of 5 min of vibration stress (gray bar) on tail flick latency (TFL) in female rats that had undergone 7 days treatment with progesterone (HP) or 6 days treatment and 24 h progesterone withdrawal (PWD). Control groups were not exposed to vibration stress. All values (mean±SEM) are expressed as a percentage of mean prestress baseline values (n=5–6 per group). ^**p<0.01, post hoc Dunnett's test in comparison to baseline readings after significant (p<0.05) repeated measures one-way ANOVA applied to raw data. F(8,5,40)=4.044, p=0.001.

Figure 8

Mean density of Fos-like immunoreactive nuclei in the whole periaqueductal gray matter (PAG) of rats in the stressed and control groups undergoing progesterone treatments (n=6 per group) Abbreviations as in Table 1. All values mean±SEM; ^*p<0.05. ^**p<0.01, ^***p<0.001, post hoc Bonferroni test after significant (p<0.05) one-way ANOVA. F(3,19)=8.700, p=0.008.

Figure 9

Effect of exposure to 5 min vibration stress on the density of Fos-immunoreactive cells in individual columns of the periaqueductal gray matter (PAG) at rostral (levels I and II) and caudal (levels III and IV) levels in rats undergoing withdrawal from progesterone. Cartoon depicts areas of PAG sampled (for more details see Figure 2). Histograms show mean Fos immunoreactive cell density in different regions of the PAG. All values mean±SEM; ^*p<0.05, ^**p<0.01, post hoc Bonferroni test in comparison to the control group after significant (p<0.05) one-way ANOVA. DL: F(3,20)=6.726, p=0.0026; LI: F(3,20)=5.437, p=0.007; LII: F(3,20)=5.181, p=0.082; VL: F(3,20)=6.515, p=0.003.