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Maguire JL, Stell BM, Rafizadeh M, Mody I
Nat Neurosci 2005;8(6):797–804 [PubMed]
Disturbances of neuronal excitability changes during the ovarian cycle may elevate seizure frequency in women with catamenial epilepsy and enhance anxiety in premenstrual dysphoric disorder (PMDD). The mechanisms underlying these changes are unknown, but they could result from the effects of fluctuations in progesterone-derived neurosteroids on the brain. Neurosteroids and some anxiolytics share an important site of action: tonic inhibition mediated by δ subunit–containing GABAA receptors (δGABAARs). Here we demonstrate periodic alterations in specific GABAAR subunits during the estrous cycle in mice, causing cyclic changes of tonic inhibition in hippocampal neurons. In late diestrus (high-progesterone phase), enhanced expression of δGABAARs increases tonic inhibition, and a reduced neuronal excitability is reflected by diminished seizure susceptibility and anxiety. Eliminating cycling of δGABAARs by antisense RNA treatment or gene knockout prevents the lowering of excitability during diestrus. Our findings are consistent with possible deficiencies in regulatory mechanisms controlling normal cycling of δGABAARs in individuals with catamenial epilepsy or PMDD.
Shen H, Gong QH, Yuan M, Smith SS
Neuropharmacology 2005;49(5):573–586 [PubMed]
In this study, 48-h administration of 3α-OH-5β-pregnan-20-one (3α,5β-THP) or 17β-estradiol (E2)+progesterone (P) to female rats increased expression of the δ subunit of the GABAA receptor in CA1 hippocampus. Coexpression of α4 and δ subunits was suggested by an increased response of isolated pyramidal cells to the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), following 48-h steroid treatment, and nearly complete blockade by 300 μM lanthanum (La3+). Because α4βδ GABAA receptors are extrasynaptic, we also recorded pharmacologically isolated GABAergic holding current from CA1 hippocampal pyramidal cells in the slice. The La3+-sensitive THIP current, representative of current gated by α4βδ GABAA receptor, was measurable only following 48-h steroid treatment. In contrast, the bicuculline-sensitive current was not altered by steroid treatment, assessed with or without 200 nM gabazine to block synaptic current. However, 48-h steroid treatment resulted in a tonic current insensitive to the benzodiazepine agonists lorazepam (10 μM) and zolpidem (100 nM). These results suggest that 48-h steroid treatment increases expression of α4βδ GABAA receptors, which replace the ambient receptor population. Increased anxiolytic effects of THIP were also observed following 48-h steroid treatment. The findings from the present study may be relevant for alterations in mood and benzodiazepine sensitivity reported across the menstrual cycle.
GABAA receptors are the primary mediators of inhibitory signaling in the brain, making them important targets for therapeutic drugs. One such class of GABAA receptor modulators, the neurosteroids, can be either excitatory or inhibitory, depending on the particular steroid and GABAA receptor isoform (1,2). Classically, steroid hormones are thought to regulate gene transcription by interacting with nuclear hormone receptors. In contrast, neurosteroids interact directly with GABAA receptors independently of the nuclear hormone receptors (3), and, therefore, research has focused on the acute effects of neurosteroids on brain physiology. Chronic treatment with other GABAA receptor modulators, such as benzodiazepines or alcohol, causes a state of behavioral tolerance, in part as a result of altered GABAA receptor expression (4,5). Although tolerance is less likely to occur with neurosteroids, prolonged neurosteroid administration does alter the function and expression of GABAA receptors (6). Endogenous neurosteroid synthesis occurs in the periphery as well as within the brain. Shifts in neurosteroid production are evident over the course of the ovulatory cycle. The studies presented here explore the exciting possibility that, in addition to their well-known and immediate modulation of GABAA receptors, neurosteroids may have long-term effects that regulate inhibitory neurotransmission.
Women of reproductive age exhibit dramatic changes in ovarian hormones during the course of the menstrual cycle. Following the midcycle hormone surge and subsequent ovulation, there is a period of 1 to 2 weeks in which the corpus luteum produces large amounts of progesterone (luteal phase). As the corpus luteum begins to fail, progesterone levels fall, thereby signaling the onset of menses and the end of the reproductive cycle. Progesterone has acute sedating and antiepileptic properties, largely as a result of its conversion to neurosteroids via the actions of 3α-hydroxysteroid dehydrogenase and 5α-reductase in various tissues. The abrupt decline in progesterone/neurosteroids is thought to be one of the reasons that many women with epilepsy have increased seizure frequency immediately prior to menses.
In an effort to clarify the relationship between menstrual changes and seizure frequency, hormonal manipulations to rodent models of epilepsy are often used. However, there are important differences between the ovulatory cycles of rodents and primates. In rats, a hormone surge occurs on the day of proestrus, followed by ovulation and a period of sexual receptivity (estrus) that is accompanied by low estrogen and progesterone levels. Then, similar to the primate luteal phase, there is a brief period of rising progesterone (diestrus). However, unlike the human luteal phase, diestrus lasts for only 2 to 3 days; thus, the cycling rodent does not have the prolonged period of progesterone exposure, which is a key feature of the primate menstrual cycle.
Some rodent models of chronic progesterone have included “pseudopregnancy” or administration of exogenous progesterone to more closely model premenstrual changes in women (2). Although acutely administered progesterone and neurosteroids are sedating, prolonged progesterone exposure in some models results in a brief period of increased anxiety behavior and benzodiazepine resistance. After a few days, these changes resolve, but subsequent progesterone withdrawal causes a rebound hyperexcitable state, with increased anxiety and propensity toward seizures. These changes are not due to progesterone itself, but rather to its conversion to neurosteroids. Chronic progesterone treatment results in elevated levels of 3α-hydroxy-5α-pregnan-20-one (3α, 5α-THP), or allopregnanolone, in the blood and CNS (7). Furthermore, prolonged application of either allopregnanolone or 3α, 5β-THP (pregnanolone) mimics the effects of chronic progesterone. Finally, inhibition of 3α-hydroxysteroid dehydrogenase or 5α-reductase, which prevents the conversion of progesterone to allopregnanolone, abolishes the effects of chronic progesterone exposure.
The cellular basis for this neurosteroid-induced hyperexcitable state remains unknown. However, subacute (2 to 4 days) neurosteroid exposure in rats is associated with increased hippocampal α4 GABAA receptor expression, which returns to baseline levels with prolonged neurosteroid treatment. On neurosteroid withdrawal, there is a powerful rebound increase in α4 GABAA receptor subunit expression. The hyperexcitability induced by both subacute neurosteroids and neurosteroid withdrawal can be blocked with intracerebroventricular injection of antisense α4 mRNA. During these hyperexcitable states, inhibitory postsynaptic currents (IPSCs) in CA1 neurons are shortened in duration and are benzodiazepine insensitive yet can be enhanced by the α4/6βγ-selective imidazobenzodiazepine compound Ro 15–4513. Since recombinant α4β2γ2 GABAA receptors deactivate more quickly than those containing α1β2γ2, it has been suggested that synaptic α1βγ GABAA receptors may be replaced by α4βγ GABAA receptors during hyperexcitable states (8).
In addition, α4 proteins can combine with other GABAA receptor subunits, such as the δ subunit. Neurosteroids (including pregnanolone and allopregnanolone) are especially powerful modulators of αβδ-containing GABAA receptors. These neurosteroids increase the maximal GABA-evoked current several fold and convert αβδ GABAA receptor responses to highly desensitizing, slowly deactivating currents (9). Furthermore, in some brain regions, the αβδ-containing GABAA receptors are thought to reside outside the synapse where they are nearly continuously exposed to low levels of GABA. Although, the tonic currents conveyed by this population of GABAA receptors are small, their long duration allows them to convey a very large overall charge transfer. The studies presented here test the hypothesis that progesterone/neurosteroid exposure alters inhibitory signaling in the CNS by regulating the expression of αβδ-containing GABAA receptors in specific brain regions.
The paper by Shen et al. explores the effects of subacute (2 days) progesterone or neurosteroid treatment on GABAA receptor expression in CA1. These studies found increased levels of α4 and δ proteins, with more modest reductions in the expression of α1 and γ2 proteins. Since αβδ GABAA receptors convey the tonic current in some brain areas, the authors tested the hypothesis that steroid treatment would alter the tonic current in CA1 hippocampal neurons. Although neurosteroid treatment did not alter amplitude of the tonic current, there did appear to be a change in the relative contribution of different GABAA receptor isoforms. The CA1 tonic current in control rat brain slices is largely conveyed by α5βγ GABAA receptors (10). So application of benzodiazepine-like compounds, including lorazepam (enhances α1,2,3,5βγ), zolpidem (enhances α1βγ and to a lesser extent α2,3βγ), and Ro 15–4513 (enhances α4,6 βγ), were used to test for the relative contribution of various αβγ GABAA receptors. Unlike control slices, the tonic currents from neurosteroid-treated rats were insensitive to all three agents, suggesting that the contribution of all γ-containing GABAA receptors to the tonic current was reduced. 4,5,6,7-Tetrahydroisoxazolo-[5,4-c]-pyridine-3-ol (THIP), also known as gaboxadol, is a partial agonist at most GABAA receptors. However, this compound is a superagonist at α4βδ GABAA receptors, with a maximal response that is greater than the response to GABA. Following neurosteroid treatment, the whole cell currents from steroid-treated cells were more sensitive to THIP than GABA. Furthermore, although the trivalent heavy metal lanthanum (La3+) interacts with several different receptors and ion channels, its effects on GABAA receptors are relatively selective. While La3+ often enhances GABAA receptor currents, low concentrations of La3+ act as a selective inhibitor of α4βδ GABAA receptors. As might be expected with increased α4βδ GABAA receptor expression, the large THIP-induced current was almost completely blocked by La3+, while cells from control slices were relatively insensitive. Taken together, the authors make a compelling argument that subacute neurosteroid treatment alters the composition of GABAA receptor subtypes that convey the tonic current in hippocampal neurons.
The paper by Maguire et al. takes the findings of Shen and colleagues a step further and explores the regulation of GABAA receptor expression over the ovulatory cycle. This study found that hippocampal δ GABAA receptor subunit expression in mice is increased and γ2L GABAA receptor expression is reduced during a period of elevated progesterone (late diestrus), compared with hippocampal GABAA receptor subunit expression during a low-progesterone period (estrus). Unlike exogenous neurosteroid treatment, α4 GABAA receptor expression was not altered. To explore the physiologic significance of these changes, electrophysiological recordings were made in hippocampal brain slices taken from mice during estrus and compared with those from mice in late diestrus. There was no difference in the amplitude, frequency, or decay time of IPSCs in either CA1 or dentate granule neurons. Moreover, similar to the finding by Shen et al., there was no change in tonic current amplitude in CA1 neurons. However, in dentate granule cells, in which the tonic current is largely conveyed by αβδ GABAA receptors (11), the amplitude of the tonic current was enhanced during diestrus. These cellular changes were associated with behavioral evidence of reduced anxiety and a lower propensity for seizures. Altered δ expression appeared to play a causative role in these changes, as there was increased sensitivity to the antiepileptic effects of THIP. Furthermore, experiments with intracerebroventricular injection of antisense δ mRNA or using δ knockout mice confirmed that the tonic current in dentate gyrus granule cells is carried largely by αβδ-containing GABAA receptors, and that enhanced expression of the δ subunit is required for the seizure resistance seen during diestrus.
A strength of the paper by Maguire and colleagues is that it addresses GABAA receptor function as it relates to the reproductive cycle. However, control of the ovulatory cycle involves complex interactions among hormones and CNS function. It should be noted that other than measuring blood progesterone levels, the Maguire et al. study did not specifically test whether the observed changes were actually caused by progesterone or neurosteroids. Since both the Maguire and Shen et al. studies used reproductively intact animals, it is entirely possible that the altered GABAA receptor expression in cycling animals is independent of progesterone/neurosteroids and involves some other component of the reproductive axis. The involvement of another regulatory system may explain some of the discrepancies in the findings between the two papers. In contrast to the Shen et al. study, the cycling mice utilized in the Maguire et al. study did not have the elevated α4 GABAA receptor expression, shortened IPSCs, or behavioral hyperexcitability seen during subacute neurosteroid treatment in rats.
An alternate interpretation of these discrepant findings is that the concentration and duration of progesterone/neurosteroid exposure may be crucial in regulating GABAA receptor expression. It is unclear how long the progesterone levels were elevated in the mice described in the Maguire paper, but previous hormone profile studies would suggest that the exposure probably only lasted a few hours to a day. During prolonged neurosteroid exposure and withdrawal, there is also increased expression of α4 containing GABAA receptors. Elevated expression of both α4 and δ GABAA receptor subunits could certainly alter the relative proportion of α1βδ-, α4βδ-, α1βγ-, and α1βγ-containing GABAA receptors in the hippocampus. GABAA receptors containing α4βδ subunits are sensitive to low levels of GABA, and α4βγ GABAA receptors deactivate quickly after brief GABA applications. However, the response of these receptors to prolonged or repetitive GABA exposure remains unknown, and so it is unclear how the transient hyperexcitable state caused by subacute neurosteroid treatment and withdrawal is actually related to altered GABAA receptor expression.
Maguire et al. associate the increased δ subunit expression in cycling mice with enhanced tonic current amplitude and seizure resistance. However, it should be kept in mind that δ-containing GABAA receptors are particularly sensitive to modulation by neurosteroids. It is unlikely that residual gonadal steroids are present in the brain slices used for electrophysiological recordings. In contrast, it is possible that the local production of neurosteroids continues in hippocampal slices and that the capacity to synthesize neurosteroids varies over the estrous cycle, thereby contributing to the altered tonic current measured by Maguire et al. Future studies will need to determine whether modulation of neurosteroid synthetic enzymes allow a level of control over CNS excitability throughout the ovarian cycle.
The studies presented here show that in addition to acute allosteric effects, neurosteroids may regulate CNS excitability by altering the expression of their target GABAA receptors. It will be important to further define the time course and regional specificity of neurosteroid regulation of GABAA receptor expression. Hormone levels change quickly over the reproductive cycle, and the response to those hormones may be equally fluid. Just as important, many of the changes in GABAA receptor expression described here are similar to those seen in some models of epilepsy. An active area of research involves exploring the possibility that similar regulation of certain GABAA receptor isoforms is involved in epileptogenesis. The studies reviewed here help highlight the fine temporal and spatial specificity of CNS excitability and provide insights into the exquisite choreography controlling the ballet of brain function in health and disease.