# The Role of the Periaqueductal Gray in  the Modulation of Pain in Males and Females: Are the Anatomy and Physiology Really that Different?

Authors: Dayna R. Loyd; Anne Z. Murphy
Journal: Neural Plasticity
 (2009)

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## Abstract

Anatomical and physiological studies conducted in the 1960s identified the periaqueductal gray (PAG) and its descending projections to the rostral ventromedial medulla (RVM) and spinal cord dorsal horn, as a primary anatomical pathway mediating opioid-based analgesia. Since these initial studies, the PAG-RVM-spinal cord pathway has been characterized anatomically and physiologically in a wide range of vertebrate species. Remarkably, the majority of these studies were conducted exclusively in males with the implicit assumption that the anatomy and physiology of this circuit were the same in females; however, this is not the case. It is well established that morphine administration produces greater antinociception in males compared to females. Recent studies indicate that the PAG-RVM pathway contributes to the sexually dimorphic actions of morphine. This manuscript will review our anatomical, physiological, and behavioral data identifying sex differences in the PAG-RVM pathway, focusing on its role in pain modulation and morphine analgesia.


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## Body

## 1. Introduction

It  was first  reported that electrical
stimulation of the midbrain periaqueductal gray (PAG) produced profound
analgesia in the male rat in 1969 [1]. Since then, the anatomical and
physiological organization of the PAG and its descending projections to the
rostral ventromedial medulla (RVM) and dorsal horn of the spinal cord have been well
characterized in a variety of species, including the rat [2–9], cat [10–18], primate [19, 20], and rabbit [21] (see Figure 1). The PAG-RVM-spinal
cord pathway comprises an essential neural circuit for opioid-based
antinociception [6, 18, 22]. Intra-PAG
administration of the mu opioid receptor (MOR) agonist morphine, the most commonly
prescribed opiate for persistent pain relief, produces naloxone-reversible
analgesia [23] as well as naloxone-reversible
excitation of RVM neurons [7, 24, 25]. Similarly,
lesions of the PAG or intra-PAG administration of MOR
antagonists [26–29] attenuate the antinociceptive
effects of systemic morphine across a wide range of analgesiometric tests [30]. Studies utilizing
autoradiography, immunohistochemistry, and in situ hybridization have shown
that the PAG contains a high density of MOR [31–38], with
approximately 27–50% of PAG
neurons retrogradely labeled from the RVM expressing MOR [35, 37].Figure 1
A
schematic of the descending inhibitory pathway for pain modulation illustrating
the projections from the midbrain periaqueductal gray to the brainstem RVM and the spinal
cord dorsal horn.While it is
well established that the PAG-RVM-spinal cord pathway is essential for the
analgesic actions of both systemic and intra-PAG morphine, these early studies
were conducted exclusively in male subjects. Only recently have studies begun
including “sex” as an independent variable, and it is becoming increasingly
clear that morphine does not produce the same degree of antinociception in males
and females, especially following the induction of persistent pain. Sex
differences in morphine potency were first reported in rodents in the late
1980s, when it was shown that systemic morphine administration produced a
significantly greater degree of antinociception in males using acute pain
assays [39–42]. This
phenomenon has been repeated in multiple studies employing animal models of
pain, including orofacial [43] and visceral [44, 45] pain models, as well as persistent
somatic pain models [38, 46–52]. Although
results on the contrary are also reported, generally these studies have shown
that morphine produces a significantly greater degree of analgesia in males in
comparison to females. Indeed, we have recently reported that male rats have a significantly higher MOR expression in the PAG, which is positively correlated with morphine analgesia in male but not female rats [38].Recently,
clinical studies in humans have also reported sex differences in morphine
analgesia. Of the limited number of studies that examined “gender” or “sex” as
an independent variable, it has been reported that males experience greater
morphine analgesia compared to females [53–55]. In fact, one
study reported that females required 30% more morphine to reach the same level
of analgesia as males [55]. Similar to the rodent literature, the
results in human studies are not unequivocal. Sarton et al. [56] reported greater morphine analgesia in
females, while two studies reported no sex difference [57, 58]. Sex
differences in morphine consumption also have been reported [59]; however, given that the
majority of negative side effects associated with morphine consumption,
including nausea, headache, and dysphoria [57, 60], are
exacerbated in females compared to males, morphine consumption is not a
reliable indicator of morphine analgesia.Sex differences
in opioid analgesia are not limited to mu opioid agonists. In both human and
animal studies, sex differences in the analgesic effects of kappa or delta
opioid agonists have also been reported, although again, not without
controversy [61–65]. Several
factors are likely to contribute to the disparate results between studies
reporting the presence or absence of a sex difference in opioid analgesia,
including differences in the type of pain being examined (e.g., experimental
acute pain versus postoperative pain versus a chronic pain state), the route of
drug administration (e.g., oral versus intravenous versus intrathecal), the strain
differences in the rodents studies, and the efficacy of the opiate being
administered. Sex differences in basal pain sensitivity, as well as estrous
cycle effects, may also contribute [54, 55, 57, 66–79].While it is
clear that sex differences in opioid analgesia are not a simple and straightforward
phenomenon, when sex differences are reported, they are not trivial in
magnitude. In our persistent inflammatory [46] and visceral pain [38, 44, 45] studies, the ED50 for
females is twice the ED50 of males. Similarly, morphine is
approximately 5-fold more potent in producing antihyperalgesia in arthritic
males compared to arthritic females [52]. Sex differences
in morphine analgesia are not due to sex differences in the pharmacokinetics of
morphine in humans [56] or rodents [50]. Rather, sex differences in morphine
analgesia are likely related to the inherent differences in how the central
nervous system of males and females respond to opiates. To date, the mechanism(s)
underlying the sexually dimorphic actions of morphine remain unknown.Given that the
PAG and its descending projections to the RVM and dorsal horn of the spinal
cord provide a primary pathway for the actions of opiates in pain modulation,
inherent differences in this pathway could contribute to the sexually dimorphic
actions of morphine. Thus, we tested three hypotheses: (1) are there sex
differences in the anatomical organization of the PAG-RVM pathway? (2) is there
a sexually dimorphic response of the PAG-RVM output neurons to persistent pain?
(3) does the administration of morphine differentially engage the PAG-RVM
pathway in male and female rats?


## 2. Sexually Dimorphic Organization of a Descending Pain Inhibitory Pathway

We used
neuroanatomical tract-tracing techniques to examine whether there were
qualitative and/or quantitative differences in the neural projection from the
PAG to the RVM in male and female rats. Consistent with previous anatomical
studies [2, 80, 81], we reported
that the dorsomedial, lateral and ventrolateral PAG heavily project to the RVM
in both male and female rats [82]. While no qualitative sex
differences were noted in the overall distribution of PAG-RVM projection neurons,
females had significantly more PAG-RVM output neurons across the rostrocaudal
axis of the PAG compared to males [83, 84] (Figures 2(a)–2(c)). The average
number of retrogradely labeled cells across the rostrocaudal extent of the PAG
was greater by a third in female compared to male rats (Figure 3(a)). The most
prominent sex difference in retrograde labeling was observed within the lateral
and ventrolateral regions of the PAG, an area known to contain a dense distribution
of MOR [34, 37].(a)–(c) Distribution
of cells retrogradely labeled (FG+) from the RVM in males
(left) and females (right) at three representative rostrocaudal levels of the
periaqueductal gray. Each black circle represents one FG+ cell. (d)–(f) Distribution
of PAG cells that were immunoreactive for AR (closed circles) or ERα (open circles). (g)–(i) Distribution
of PAG cells retrogradely labeled from the RVM that
were also immunoreactive for AR (closed stars) or ERα (open stars).Figure 3
(a) Mean
number of PAG cells retrogradely labeled from the RVM
across the rostrocaudal axis in males (left) and females (right). (b) Percentage of Fos-positive neurons
that were retrogradely labeled from the RVM in males (left) and females (right)
following twenty-four hours of inflammation. (c) Average of the percentage of AR (left) and ERα
(right) receptor-expressing PAG cells retrogradely labeled from the
RVM. (d) Percentage of Fos-positive neurons that were retrogradely labeled from the RVM
in males (left) and females (right) following twenty-four hours of inflammation
and one hour of morphine (5 mg/kg).


## 3. Sexually Dimorphic Response of the PAG-RVM Pathway to Persistent Inflammatory Pain

Inflammatory pain results in the
activation of descending modulatory circuits [8, 85] and
contributes to both hyperalgesia and antinociception [86–89]. We found
that the persistent inflammatory  pain induced by injection of complete Freund's adjuvant (CFA) into the rat
hindpaw caused extensive activation of PAG neurons as measured by Fos labeling.
Interestingly, this activation was comparable (both quantitatively and
qualitatively) in male and female rats [82]. However, when the analysis was
restricted to PAG neurons retrogradely labeled from the RVM, while females have
almost twice the number of PAG-RVM output neurons in comparison to males, very
few of these cells in female rats expressed inflammation-induced Fos,
suggesting that this circuit is preferentially activated in males (Figure 3(b)).
Indeed we found that, overall, persistent inflammatory pain activated
approximately 43% of PAG-RVM neurons in the dorsomedial, lateral and
ventrolateral PAG of males, but only half as many PAG-RVM output neurons were
activated by inflammatory pain in females. Activation of the PAG and its
descending outputs to the RVM results primarily in the inhibition of dorsal
horn neuronal responses to acute noxious stimuli [90–95]; therefore,
one would predict that given the greater activation of the circuit in males
than females, males should have displayed reduced hyperalgesia following
induction of plantar inflammation. However, in our behavioral studies, we found
no sex differences in either baseline withdrawal latencies or in CFA-induced
hyperalgesia. Therefore, our finding that the PAG-RVM descending circuit is not
being engaged to the same degree by persistent inflammatory pain in males and
females suggests that there is an alternative mechanism for endogenous pain
modulation in female rats [96–99].We have recently begun exploring this
possibility using combinatorial anterograde and retrograde tract-tracing in
combination with persistent pain-induced Fos labeling. The results of these
studies suggest that there are indeed sex differences in both the efferent and
afferent projections of the PAG. Specifically, the amygdala, ventromedial
hypothalamus, and periventricular nucleus project more heavily to the PAG in
females than males. In contrast, the medial preoptic area, parabrachial
nucleus, and locus coeruleus project more heavily to the PAG in males than
females [100]. In addition,
our data indicate that the projections to the parabrachial nucleus, locus
coeruleus, and the A5/A7
noradrenergic cell group appear to be greater in males (Loyd and Murphy,
unpublished observations). Obviously, further research on the anatomy and
physiology of pain modulatory circuits in females is warranted.


## 4. Sex Differences in the Activation of the Descending Inhibitory Pathway by Morphine

Although sex
differences in PAG-RVM output neuron activation do not appear to contribute to
sex differences in pain, they do appear to contribute to sex differences in
morphine analgesia. Until recently, all studies examining the mechanisms of
morphine action in the PAG were conducted exclusively in males; therefore it
was unknown whether morphine administration has the same physiological effect
on PAG neurons in females. Electrophysiological studies of PAG neurons are
limited because they examine the response of a single neuron [7, 17, 101–109]. We have
addressed this problem by using tract-tracing techniques and Fos labeling to
measure the activity of populations of PAG-RVM neurons in the PAG of males and females.Systemic
morphine administration attenuates the persistent pain-induced Fos expression
within the PAG of male but not female rats [82] and is
consistent with our data showing that the ED50 for systemic morphine
is approximately twofold higher in females compared to males whether administered systemically [46] or directly into the PAG [38].
Interestingly, morphine administration, in the absence of pain, resulted in a
twofold greater activation of PAG neurons compared to saline administration [84]. No sex difference was observed
in the activation of PAG neurons by morphine (see the black circles in Figures
4(a)–4(c)), suggesting
that in the absence of pain, morphine is equipotent in its ability to
depolarize PAG neurons. However, when the analysis was limited to PAG neurons
projecting to the RVM, the number of neurons activated by morphine was
consistently and significantly higher in males compared to females (see the
stars in Figures 4(a)–4(c)) [84]. Indeed, approximately half of
PAG-RVM neurons in males were activated by morphine, whereas only 20% were
activated in females (see Figure 3(c)). These results corroborate previous
studies demonstrating that morphine results primarily in the net excitation of
PAG-RVM neurons, most likely through the removal of tonic GABA inhibition [35, 104, 110, 111]. The finding that very few
PAG-RVM neurons were activated by morphine in females suggests that morphine
may be limited in effectiveness as a pain modulator.Distribution
of PAG cells expressing Fos (black circles) and cells retrogradely labeled from
the RVM expressing Fos (stars) following: (a)–(c) one
5 mg/kg dose of morphine; (d)–(f) one
5 mg/kg dose of morphine per day for three consecutive days; (g)–(i) or two
5 mg/kg doses of morphine per day for three consecutive days in males (left) and
females (right) at three representative rostrocaudal levels of the
PAG.Given that more PAG neurons
project to the RVM in female compared to male rats, it is possible that pain
modulation in females is less dependent on opioids. If this is the case, then
direct activation of PAG output neurons should produce greater antinociception
in females, not males. Microinjection of the GABA antagonist bicuculline into
the PAG produces antinociception [110, 112] by
disinhibiting output neurons. Surprisingly, even though females have more
output neurons, the antinociceptive effect of microinjecting bicuculline into
the PAG is greater in males [113].


## 5. Sex Differences in the Development of Tolerance to Morphine

Repeated or
continuous administration of morphine into the ventrolateral PAG of male rats
has been shown to result in the development of tolerance [26, 114–118]. In addition, blocking opioid
binding sites in the ventrolateral PAG attenuates the development of tolerance
to systemically administered morphine [26]. Tolerance appears to be
mediated by a reduction in MOR signaling efficacy in PAG neurons
[119], an effect
that is reversed when MOR coupling is enhanced via upregulated
adenylate cyclase activity [120]. If the
PAG-RVM pathway is essential for the development of tolerance, then activation
of the PAG-RVM pathway by morphine should decline as tolerance develops, and
changes in the activation of this pathway would correlate with sex differences
in the development of tolerance to morphine. These hypotheses were tested in
male and female rats using behavioral testing (hot plate) and
immunohistochemistry to map the activation of the PAG-RVM pathway following
repeated morphine administration.Morphine was administered once or twice a day
for three days in rats that had previously received retrograde tracer
injections into the RVM. To examine the activation of PAG-RVM neurons during
the development of tolerance, males and females were both administered 5 mg/kg of morphine, the ED50 for males.
Repeated administration of  systemic morphine induced tolerance in males to a
significantly greater extent than in females [83], consistent
with previous research administering equipotent doses of morphine to examine
sex differences in tolerance [47]. The half maximal
antinociceptive effect of a single injection of morphine following the
development of morphine tolerance was two times greater for female compared to
male rats. In parallel, the activation of PAG-RVM neurons was significantly
attenuated following repeated morphine administration in males [83]. While there
was no sex difference in the activation of the PAG following three doses or six
doses of morphine over three days (see the black circles in Figures 4(d)–4(i)), the
activation of the PAG-RVM projection neurons steadily declined in males only (see
the stars in Figures 4(d)–4(i)). Activation
of the PAG-RVM pathway by morphine in female rats was minimal, and therefore
did not decline significantly following repeated administration of morphine
(Figure 5; previously published [83]).Percentage
of Fos-positive neurons that were retrogradely labeled from the RVM (%Fos in
FG+ cells) in male (solid bars) and female (open bars) rats injected with
either morphine or saline once or twice daily for three days for the rostral
((a); Bregma −6.72, −7.04, −7.74) and caudal ((b); Bregma −8.00, −8.30, −8.80) PAG.
A decrease in labeling is evident with an increase in the number of morphine
injections for male rats. The # indicates a significant effect of treatment and
the * indicates a significant effect of sex.Saline: morphine naïve; 1 dose: saline pretreatment followed by one
dose of morphine; 3 doses: one dose of morphine per day; 6 doses: two doses of
morphine per day.
(a)
Rostral PAG(b)
Caudal PAGWhile
together, these data provide compelling support for a central role of the PAG
in the development of morphine tolerance; these studies administered themale ED50 dose of morphine. While a single
administration of this dose of morphine resulted in comparable activation of
the PAG in males and females, it was suboptimal in producing behaviorally defined
antinociception in females and may account for why females did not develop
tolerance to the same degree as males. Future studies employing sex-specific ED50 doses are
clearly warranted.


## 6. Role of Gonadal Hormones in Sex Differences in Morphine Analgesia

Studies in
rodents indicate that sex differences in the organizational and activational
effects of the gonadal hormones estradiol and testosterone influence morphine
analgesia. For example, male rats castrated at birth demonstrate decreased
morphine potency in adulthood, while female rats masculinized at birth
demonstrate greater morphine potency in adulthood [121, 122]. Similarly, morphine is less
effective in gonadectomized adult males and is more effective in ovariectomized adult females [40, 123–128]; these effects can be reversed
with hormone replacement [44, 123, 129]. Moreover, the antinociceptive
potency of morphine has been reported to be greater during diestrus, when
circulating estradiol levels are lowest [43, 124, 125, 127, 130], which is corroborated by our recent findings that MOR expression in female rats is the highest during diestrus compared to proestrus and estrus [38]. Recently, it was reported that
microinjection of morphine directly into the PAG produces less antinociception
during estrus (after estradiol peaks), while there was no sex difference in
morphine potency between diestrus females and males [131]. We have
recently reported similar findings in which the antihyperalgesic effects of
intra-PAG morphine were significantly greater in females in diestrus in comparison to proestrus and estrus [38].The anatomical substrate(s) whereby
gonadal steroids influence pain and analgesia is unknown. Both androgen (AR) and estrogen
receptorsα (ERα) have been localized in the PAG in the male
rat [132]. Although it is not known if these
receptors are present in the female rat, they have been localized in other
species including the female cat [12], golden
hamster [133], guinea pig [134], and the rhesus monkey [135, 136]. To date,
however, the anatomical distribution of both types of steroid receptor within
the PAG in reference to cells projecting to the RVM is not known.We have
combined neuroanatomical tract-tracing techniques and steroid receptor
immunohistochemistry to characterize the  expression of AR and ERα in the PAG-RVM pathway of male and female rats
[137]. In these studies, we found that
males had a significantly greater number of AR immunoreactive neurons localized
within the dorsomedial, lateral and ventrolateral PAG compared to females.
Interestingly, both the qualitative and quantitative expression of ERα in the PAG was comparable between the sexes (see
Figures 2(d)–2(f)). Both
receptor types were preferentially localized within the dorsomedial, lateral
and ventrolateral subdivisions of the PAG and increased in density along the
rostrocaudal axis of the PAG with the highest expression localized within the
caudal PAG. In
addition, 30–37% of PAG-RVM
output neurons expressed AR or ERα (Figure 3(d)) with the highest density of
colabeling in the lateral/ventrolateral region of PAG. ERa and AR
colocalization in PAG  neurons projecting to the RVM was comparable between the
sexes [137] (Figures 2(g)–2(i)). The
high density of steroid receptors localized on PAG-RVM output neurons may contribute to our observed sex differences in morphine
analgesia. Although there was no sex difference in the anatomical
localization of gonadal steroid receptors in the PAG despite the higher density
of AR in males, 27–50% of PAG-RVM
neurons contain  MOR [37]. Given that morphine activates
more of these neurons in male compared to female rats, the interaction between
morphine and sex hormones is likely greater in the PAG of male compared to
female rats.There are
several mechanisms whereby gonadal steroids may modulate opioid-sensitive
PAG-RVM output neurons, thereby potentially resulting in a dimorphic response
to morphine. First, estradiol has been shown to
uncouple the MOR from G protein-gated inwardly rectifying
potassium channels [138] resulting in an
attenuation of morphine-induced hyperpolarization. Second, estradiol has also
been shown to induce MOR internalization [139], thereby reducing
available opioid binding sites on the cell membrane. Interestingly, ERα is required for estradiol-induced MOR
internalization [140] supporting
the hypothesis that colocalization of MOR and ERα in the PAG-RVM output neurons may provide a pain
modulatory mechanism. Interestingly, administration of estradiol to
gonadectomized males reinstates morphine analgesia while dihydrotestosterone
does not [141], suggesting that estrogens
affect morphine potency in both male and female rats [130].


## 7. Conclusions

Research
spanning for four
decades has shown that the PAG and its descending projections to the RVM and
spinal cord dorsal horn constitute an essential neural circuit for opioid-based
analgesia. During the last half of that period, numerous rodent and human
studies have established sex differences in the antinociceptive and analgesic
effects of morphine; however, the neural mechanisms underlying the sexually
dimorphic actions of morphine remain poorly understood. It is now clear that
the anatomical and physiological characteristics of the PAG and its descending
projections to the RVM are sexually dimorphic, with clear biological consequences
in terms of morphine potency. Our studies, as well as those of others, have
shown that morphine is less potent in females compared to males in the
alleviation of persistent pain. Future research efforts utilizing female
subjects in both the investigation of persistent pain mechanisms and
identification of both effective and potent pain therapeutics are clearly
warranted.



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