# Progesterone Withdrawal-Evoked Plasticity of Neural Function in the Female Periaqueductal Grey

Authors: T. A. Lovick; A. J. Devall
Journal: Neural Plasticity
 (2009)

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## Abstract

Cyclical changes in production of neuroactive steroids during the oestrous cycle induce significant changes inGABAA receptor expression in female rats. In the periaqueductal grey (PAG) matter, upregulation of α4β1δ GABAA receptors occurs as progesterone levels fall during late dioestrus (LD) or during withdrawal from an exogenous progesterone dosing regime. The new receptors are likely to be extrasynaptically located on the GABAergic interneurone population and to mediate tonic currents. Electrophysiological studies showed that when α4β1δ GABAA receptor expression was increased, the excitability of the output neurones in the PAG increased, due to a decrease in the level of ongoing inhibitory tone from the GABAergic interneurones. The functional consequences in terms of nociceptive processing were investigated in conscious rats. Baseline tail flick latencies were similar in all rats. However, acute exposure to mild vibration stress evoked hyperalgesia in rats in LD and after progesterone withdrawal, in line with the upregulation of α4β1δ GABAA receptor expression.


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## Body

## 1. Introduction

The periaqueductal (PAG) grey matter is involved in regulating a remarkable number
of bodily functions. Circuits controlling nociception, temperature regulation,
micturition, vocalisation, cardiorespiratory function, and sexual behaviours
are all dependent on the functional integrity of this midbrain region [1–5]. The PAG is also involved in
producing emotional changes, particularly those associated with fear and
defensive behaviour [6, 7], and has the ability to
modulate activity in its various control circuits to orchestrate changes in the
behavioural response pattern that are appropriate to an ever-changing
environment [8]. In females, the PAG operates
within a constantly changing hormonal milieu that results from the cyclical
changes in production of neuroactive gonadal steroids during the menstrual
cycle (oestrous cycle in animals). The lipophilic nature of these molecules
means that they readily gain access to the brain from the circulation [9]. Here we review the results
of our recent investigations into the functional consequences of changes in
circulating levels of progesterone during the oestrous cycle in female rats.
These experimental studies have revealed remarkable hormone-linked changes in
the intrinsic excitability of the PAG circuitry that are reflected by
significant changes in the behaviour of the animal.


## 2. Cyclical Changes in Progesterone Secretion in Females

In women, production of
progesterone undergoes substantial changes during the menstrual cycle. Plasma
levels of the steroid remain at a constant low level during the first half of
the cycle. Following ovulation, secretion of progesterone by the corpus luteum
increases, resulting in elevated blood plasma levels [10]. In the absence of a
fertilised ovum the corpus luteum then degenerates, with an associated rapid
fall in plasma progesterone production prior to menstruation.It has long been recognised that the cyclical production of
gonadal hormones during the menstrual cycle can trigger significant changes in
psychological status. In up to 75% of women, the late luteal phase of the
cycle, when progesterone levels decline rapidly, is associated with the
development of adverse psychological symptoms; these may include irritability,
mood swings, aggression, and anxiety [11]. Additionally, bodily changes
such as breast tenderness and bloating may occur and responsiveness to painful
stimuli becomes enhanced [12, 13]. Importantly, symptoms fail
to develop in women during anovulatory cycles [14] indicating a causal
relationship between changes in gonadal steroid levels and brain function. The
oestrous cycle of rodents acts as a suitable model of the human menstrual
cycle, and offers the opportunity to study hormone-induced plasticity of brain
function within intact circuits and to relate this to a behavioural outcome.
The late dioestrus (LD) phase in rats, when progesterone levels are falling
naturally, correlates with the premenstrual phase in women and increased
anxiety and aggression have been reported to develop during dioestrus in rats [15–17]. In rats progesterone levels
also fall rapidly during proestrus following the preovulatory surge [18]. However, the dynamics of
this short-lasting surge in progesterone production are not sufficient to
trigger the long-lasting changes in GABAergic function that accompany LD (see below, also [19] for discussion of this
point).


## 3. Neural Actions of the Progesterone Metabolite Allopregnanolone

Within the brain, progesterone
produces genomic effects via neuronal nuclear-bound receptors. In addition, it
can also act at the cell membrane level. These nongenomic effects are mediated
not by progesterone itself but via the actions of its neuroactive metabolite 3
alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, ALLO). ALLO is a
steroidal compound that is synthesizedde
novo within the brain from progesterone via the actions of 5α-reductase and 3α-hydroxysteroid dehydrogenase (for review, see [20]). ALLO acts at two sites on
the GABAA receptor. The first is an activation site that produces
direct receptor activation and the second is a potentiation site at which ALLO
acts as a powerful positive allosteric modulator to enhance the effects of GABA
[21]. Potentiation occurs in
response to low nanomolar concentrations of ALLO [22] whereas higher, micromolar
concentrations, as seen for example at parturition [23], are required for direct
activation of GABAA receptors [24]. The levels of ALLO present
in the brain are influenced by the circulating levels of progesterone and we
have shown recently that ALLO from the plasma gains ready access to the PAG
where it produced a decrease in neuronal excitability via a GABAA-mediated
mechanism [25]. In other brain structures,
changes in the concentration of ALLO following fluctuations in the level of
circulating progesterone have been  shown to trigger upregulation of GABAA receptor subunit expression that leads to
changes in neural excitability and behaviour [26].


## 4. Progesterone Withdrawal-Induced Plasticity of GABA Receptor Function in the PAG

GABAA receptors are pentameric structures that surround a
single chloride channel. Although most receptors are comprised of only 3 subunit types, the large pool of available subunits means that receptors can be
comprised of many different subunit combinations [27]. The
functional characteristics of individual receptor subtypes are determined by
their subunit composition. There are several indications that fluctuations in
the concentrations of progesterone play a major role in determining the
temporal pattern of expression of certain subunits of the GABAA receptor. For example, withdrawal of cultured cerebellar granule cells or
cortical neurones from long-term treatment with progesterone was accompanied by
upregulation of α4 subunit mRNA [28, 29]. Similarly,
hippocampal tissue from progesterone-withdrawn rats showed upregulation of both
α4 and δ subunit
mRNA [26, 30]. These effects were mediated not by
progesterone itself but by its neuroactive metabolite ALLO and presumably
represent a response of the neurone in an effort to maintain homeostasis. Thus
progesterone influences neural function directly via a genomic action at the
nuclear progesterone receptor and indirectly via a  nongenomic action of ALLO at
the membrane-bound GABAA receptor.The PAG is another brain region that shows a
susceptibility to phasic changes in the ambient level of progesterone and hence
ALLO. Using immunohistochemistry, we found that 24-hour withdrawal of female
rats from long-term dosing with exogenous progesterone (5 mgKg−1 IP twice daily for 6 days) leads to upregulation of α4, β1, and δGABAA receptor subunit protein in neurones in the PAG (Figure 1) [31]. Within the PAG, subunit-immunoreactive
neurones were distributed throughout all subdivisions (Figure 2). However,
upregulation after progesterone withdrawal was most marked in the dorsolateral
column [32] where the density of GABAergic neurones, in
which most α4β1δ receptors are expressed [31], was the greatest [33].Density ofα4, β1, and δGABAA receptor subunit-immunoreactive neurones
in the PAG in (a) spontaneously cycling female rats and (b) rats that had
undergone a progesterone withdrawal regime. Abbreviations: P: proestrus; O: oestrus; ED: early dioestrus; LD: late
dioestrus; HP: high progesterone; VEH: vehicle treated; PWD: progesterone-withdrawn.
n=5 for each group, all values mean±SEM. *P<.05,
post hoc Fischer
test after significant (P<.05) one-way ANOVA. Data redrawn from [31, 32].
(a)(b)Figure 2
Camera lucida reconstruction showing distribution
ofα4, β1, and δGABAA receptor subunit positive neurons in representative sections taken from the
mid PAG level from a rat in early dioestrus. The figure is adapted from [31].We have been able to translate the findings obtained using
an exogenous dosing regime to the natural fluctuations in the hormone that
occur during the oestrous cycle. During LD, when plasma levels of
progesterone are falling, a parallel upregulation ofα4, β1, and δGABAA receptor subunit protein occurred within the PAG [34]. This suggests that
new GABAA receptors  with the α4β1δ subunit configuration had
been formed in response to the falling steroid levels in the brain. GABAA receptors containing δ subunits
are likely to be extrasynaptically located [35] and those with the α4β1δ configuration are characterised by an
extremely low EC50 for GABA [36]. The α4β1δGABAA receptors in the PAG should therefore
be activated by the level of GABA present in the extracellular fluid and be
responsible for mediating tonic currents [35, 36]. Expression of α4, β1, and δ subunit
protein was confined predominantly to the GABAergic neuronal population in the
PAG [32]. These two factors, that is, sensitivity to
GABA and cellular location, may provide an important key to the functional
consequences of progesterone withdrawal-induced receptor plasticity. In
functional terms, increased expression of α4β1δ receptors on GABAergic neurones would be
expected to lead to a reduction in the level of their activity, thus
disinhibiting the output neurones within the PAG by reducing the level of
ongoing GABAergic tone (Figure 3). Hence, one would expect to see an increase
in the excitability of the various neural control systems located within the
PAG. The PAG is organised functionally in terms of a number of longitudinal
columns that integrate and control diverse aspects of its function [37]. The oestrous cycle-linked upregulation of α4, β1, and δGABAA receptor subunit protein was seen in all regions of the PAG with little
evidence of somatotopic    localisation. Hence, the functional consequences of
receptor plasticity are likely to be widespread and not confined to any one
aspect of PAG function.Cartoon to depict changes inGABAA receptor expression in the PAG at different stages of the oestrous cycle. α4β1δGABAA receptors are expressed mainly by
GABAergic interneurones where they are extrasynaptically located and mediate
tonic currents. The excitability of output neurons from the PAG is limited by
spontaneous activity in GABAergic interneurones. (a) When expression of α4β1δGABAA receptors is low during proestrus
(Pro), oestrus (O), and early dioestrus (ED), high levels of activity in the
interneurone population limit the excitability of the output neurons. (b) When
progesterone levels fall during late dioestrus (LD), increased expression of α4β1δ receptors
leads to an increase in tonic current carried by GABAergic cells, thus limiting
their on-going activity. The output neurones therefore become intrinsically
more excitable, and their threshold for activation is lowered. The figure is adapted from [38].
(a)(b)


## 5. Oestrous Cycle-Linked Changes in Neural Excitability of the Female PAG

The excitability of the neural circuits of the
PAG is normally regulated by tonic activity of GABAergic neurones. At the
neuronal level, the presence of ongoing GABAergic inhibition within the PAG was
revealed by the increase in firing rate of output neurones in the presence of aGABAA antagonist [19]. In the conscious rat, the functional
importance of the tonically active GABAergic control system is manifested by
the dramatic behavioural changes elicited by microinjection of GABAA antagonists into this region [39–42]. To date, most of these studies have been
restricted to male animals. However, given the plasticity of the GABAergic
control system in the PAG in females, changes in the functional excitability of
the neural circuitry might be expected to occur at different stages of the
oestrous cycle. Indeed, our electrophysiological studies have shown that GABA
tone in the PAG in females is reduced during LD [19] and also in oestrus, although the latter effect
is unlikely to be related to plasticity of α4β1δ receptor expression (for a discussion on this
point see [19]). Changes in the intrinsic level of GABAergic
tone in the PAG have the potential to impact significantly on the wide range of
the behaviours that are controlled by this region. Indeed, even in the
anaesthetised preparation, we were able to show changes in responsiveness of
single neurones in the PAG to the panicogenic and pronociceptive CCK2 receptor agonist pentagastrin at different stages of the cycle [19].


## 6. Behavioural Consequences of Steroid Hormone Withdrawal-Evoked Changes in PAG Function

The PAG is a source of multiple descending control pathways
to the spinal cord that exert both inhibitory and facilitatory influences on
the spinal processing of nociceptive information [43]. Both pro- and antinociceptive control systems
are thought to be tonically active under normal conditions but the balance
between them is in a state of constant dynamic flux [44, 45]. We have recently begun to investigate the
behavioural consequences  of steroid hormone-evoked changes in the excitability
of the circuitry in the conscious animal, focussing on changes in
responsiveness to painful stimuli as well as on indices of anxiety.The tail flick latency (TFL) in response to radiant heat
applied to the underside of the tail elicits a withdrawal reflex that is a
commonly used index of sensitivity to acute cutaneous pain in conscious rats [46]. We compared TFLs in rats that had undergone a
progesterone withdrawal regime (5 mg Kg−1 IP twice daily for 6 days
followed by 24-hour withdrawal) with those obtained from rats in proestrus
(Pro) and LD. These two stages of the oestrous cycle were chosen as being
representative of low and high expressions of α4β1δGABAA receptors in the PAG (see Figure 1). Since changing hormone levels might also induce changes in stress or
anxiety levels in rats used for nociception testing, which could potentially
influence their perception of pain [46], we also observed the behaviour of animals in a
1m×1m open field arena to assess intrinsic anxiety levels [47].Experiments involving nociceptive testing were carried out
under the UK Animals (Scientific Procedures) Act 1986 and
conformed with the Guide for the Care and Use of Laboratory Animals published
by the US National Institutes of Health (NIH Publication no. 85–23, revised
1985). Female Wistar rats were
habituated to a restraining tube. TFLs were measured at 5-minute intervals
over a 15-minute period, that is 3 tail flick tests, to obtain a basal value
for TFL. There was no difference in basal TFLs between any of the different
groups of spontaneously cycling rats or the progesterone-treated animals (Figure4(a)). Similarly, there was no difference in any of the behavioural indices
measured in the open field (Table 1). We next investigated interactions between
anxiety and nociception. The rats were subjected to mild nonnoxious stress by
vibrating the restraining tube with the rat inside it for 5 minutes at 4 Hz [48]. During the vibration stress, the rats showed
signs of anxiety that included micturition, defaecation, and vocalisation.
Immediately following the vibration stress, tail flick testing was resumed.
Rats that were undergoing progesterone-withdrawal had developed a poststress
hyperalgesia that was manifested as a significant decrease in tail flick
latency that persisted for 10 minutes (Figure 4(b)). Rats maintained on the
progesterone dosing schedule failed to develop hyperalgesia (Figure 4(b)).
Spontaneously cycling rats in Pro (low α4β1δGABAA receptor expression in the
PAG) were compared with rats in LD (high α4β1δGABAA receptor expression in the
PAG) using the same protocol. Rats in LD displayed a significant hyperalgesia
following the vibration stress, whereas rats in Pro showed no change in TFL
(Figure 4(b)).Table 1
Behavioural indices in open field test for rats in proestrus, late dioestrus,
and after progesterone withdrawal. All valuesmean±SEM. Late dioestrus (LD,
n=15), proestrus (Pro, n=22), high progesterone (HP, n=7).
MeasureGroupLDProHPTotal distance travelled (cm)3827±2283737±2054328±208Average speed (cms−1)12.85±0.7612.91±0.7014.45±0.70Time in central zone (%)2.56±0.511.96±0.252.82±0.56Time in middle zone (%)10.00±1.987.74±1.426.75±0.94Time in outer zone (%)86.60±2.2489.09±1.7390.85±1.43Number of central zone re-entries4.25±0.644.10±0.623.57±0.57Time rearing (%)18.53±2.3519.13±2.0114.13±1.26Time freezing (%)0.30±0.300.54±0.44n/aTime grooming (%)7.68±1.516.48±1.484.93±1.38(a)
Baseline tail
flick latency in spontaneously cycling female Wistar rats in proestrus (Pro)
and late dioestrus (LD) and in rats undergoing progesterone treatment (HP) or
withdrawal from an exogenous progesterone dosing regime (PWD). Data show mean values of three readings taken at 5-minute
intervals. All valuesmean±SEM. Pro: n=13, LD: n=12, HP: n=8, PWD: n=7. (b) Change in tail flick latency (TFL)
following 5-minute exposure to nonnoxious vibration stress in normally cycling
and progesterone-withdrawn female Wistar rats. Data represent mean values
obtained during 10 minutes immediately poststress as a percentage of the mean baseline level
measured during 10 minutes prior to the stress. A control group comprising rats
in proestrus and late dieostrus received no vibration stress. All values mean±SEM; no stress (control): n=12, Pro: n=13, LD: n=12, HP: n=8, PWD: n=7. *P<.05,
post hoc Dunnett’s test in comparison to control group after significant (P<.05)
one-way ANOVA.
(a)(b)These experiments failed to show any change in baseline
thermal nociception in female Wistar rats at different stages of the oestrous
cycle. In contrast, other investigators have been able to detect oestrous
cycle-linked differences in sensitivity to pain [49–53]. However, the reports of differences in
nociception with respect to cycle stage were equivocal. At best, it seems that
cycle stage may influence responsiveness to pain in rats but only in some
strains and under specific experimental conditions. In terms of PAG function,
this suggests either that there is very little oestrous cycle-linked change in
tonic descending control of spinal nociceptive processing or, alternatively,
that the activity in the control systems is altered during the cycle but in
such a way that there is little change in the net balance of control at the
spinal cord level.In line with the lack of oestrous cycle effect on basal pain
sensitivity, we were also unable to detect any differences in basal anxiety
levels using the open field test. This finding is supported by previous studies
[54, 55] but not by the work of Frye et al. [56] who reported an increase in anxiety-related
behaviour in a brightly lit open field during the dark phase of the day in rats
in dioestrus. However, this study may not be directly comparable to the present
work, since testing in bright light, as opposed to the relatively subdued
lighting (60 lux) used in the present study, would be inherently more stressful
to the rats [57] by compounding the anxiogenic effects of bright
light and exposure to a novel environment.Interestingly, in the present study oestrous cycle-related
differences in responsiveness to pain became apparent in the setting of a mild
stress that increased anxiety levels. Moreover, the oestrous cycle-linked
hyperalgesia appeared only during LD, whenα4β1δ receptor expression in the PAG would be
upregulated. Rats undergoing withdrawal from progesterone, in which α4β1δ receptor expression in the PAG would also be
upregulated, showed a hyperalgesia, indicating that the effect seen in the
spontaneously cycling animals was likely to be hormone-linked. During the
oestrous cycle, levels of a number of gonadal hormones change [18]. Oestradiol in particular has been shown to
affect GABAergic function [58]. However, at the time when α4β1δGABAA receptor expression increase
during LD, plasma oestradiol levels are low and stable, suggesting that changes
in progesterone rather than oestrogen are responsible for the changes in PAG
function during LD. The results of our most recent work indicate that the PAG
may be involved in mediating the oestrous cycle-linked hyperalgesia in the
setting of mild anxiety. We have found that exposure to the vibration stress
regime elicits differential expression of the immediate early gene c-fos in the ventrolateral PAG in rats in
Pro and LD (Lovick and Devall, unpublished work). The ventrolateral PAG contains neurons that are a source of descending facilitation of
spinal nociceptive inputs [59, 60]. Thus steroid hormone-linked changes in the
excitability of descending control systems from the PAG may alter the level of
descending control over spinal nociceptive processing and contribute to the
hyperalgesia that develops in LD or during progesterone withdrawal.Recent imaging studies in humans have also implicated the
PAG in anxiety-induced hyperalgesia. Anticipatory anxiety in expectation of
receiving a painful cutaneous thermal stimulus led not only to a heightened
pain experience when the stimulus was delivered, that is hyperalgesia, but was
also associated with activation of the PAG [61]. In women, increased responsiveness to noxious
stimulation has been reported consistently during the luteal phase of menstrual
cycle [12, 13]. In many women, anxiety levels are raised
during the late luteal phase [11]. In any experimental scenario involving pain
testing in human subjects, a degree of anxiety or apprehension is almost
inevitable. Thus it is possible that the reported menstrual cycle-related
differences in pain sensitivity in women may to a large extent be secondary to
changes in anxiety levels rather than a primary response to changing hormone
levels.In female rats, falling progesterone levels can produce
remarkable changes in the functional characteristics of neurones in the PAG,
which may underlie certain oestrous cycle-linked changes in behaviour. These
findings have implications for the design and interpretation of behavioural
studies in female rodents in which the stage of the oestrous cycle may be a
significant confounding influence. It is also possible that such hormone-linked
changes may underlie the development of menstrual cycle-related disorders in
susceptible women.



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