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Perimenstrual Seizures and Neurosteroid Withdrawal
Chapter 62
Perimenstrual Seizures and Neurosteroid Withdrawal Doodipala S. Reddy Texas A&M Health Science Center, Bryan, TX, United States
GENERAL DESCRIPTION The term “catamenial epilepsy” is used to describe the cyclical occurrence of seizure exacerbations during particular phases of the menstrual cycle in women with preexisting epilepsy (Herzog et al., 2008; Newmark and Penry, 1980; Reddy, 2004). The types of epilepsies and seizures that are susceptible to catamenial fluctuations are not yet thoroughly defined. However, it appears that seizures in both partial epilepsies (such as mesial temporal lobe epilepsy) and certain primary generalized epilepsies (such as juvenile myoclonic epilepsy) can exhibit catamenial exacerbations. Of note, menstrual cycle influence on seizure susceptibility has been anecdotally observed since antiquity. However, because women with epilepsies that are severe enough to exhibit cyclical changes in seizure frequency are invariably treated with antiepileptic drugs, catamenial epilepsy is now defined as a specific form of pharmacoresistant epilepsy. With standard antiepileptic drug treatment, some of these women experience a resolution of seizures except at certain times during the menstrual cycle, while others do not respond to medications at all, or display inconsistent therapeutic benefit. In either case, the subjects continue to have intractable seizures, despite the best efforts of modern medicine. Catamenial seizure exacerbations affect between 25% and 70% of women with epilepsy who are of reproductive age (Reddy, 2014). The reason for the large range is due to differences in definition; however, three types of catamenial seizures have been identified: perimenstrual (C1), periovulatory (C2), and inadequate luteal phase (C3), based on when a patient observes increases in seizure frequency/ severity (Herzog et al., 1997; Reddy, 2009) (Fig. 62.1A,B). The perimenstrual is the most common clinical type. The specific pattern of incidence can be identified simply by charting menses and seizure clusters; along with obtaining
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mid-luteal phase serum progesterone (P) levels to distinguish between normal and inadequate luteal phase cycles (Herzog et al., 2008; Quigg et al., 2009). The diagnosis of catamenial epilepsy is mainly based on the assessment of menstruation and seizure records. Using the first day of menstrual bleeding as the first day of the cycle, the menstrual cycle is divided into four phases: (1) menstrual phase, days –3 to +3; (2) follicular phase, days +4 to +9; (3) ovulatory phase, days +10 to +16; and (4) luteal phase, days +17 to –4. The number of seizures in each phase is counted for at least two cycles, and a twofold or greater increase in frequency during a particular phase of the menstrual cycle can be used as diagnostic criteria of catamenial epilepsy. In perimenstrual catamenial epilepsy (C1), women with epilepsy experience an increase in seizure activity before, during, or after the onset of menstruation (Reddy, 2009). Catamenial epilepsy is observed in women with both ovulatory and anovulatory cycles. In one study (Herzog et al., 2004), about 16.5% of subjects were found to have anovulatory cycles. These women showed a third type of catamenial epilepsy, referred to as inadequate luteal phase (C3) or anovulatory luteal seizures. Experimental models have been developed that mimic the perimenstrual seizures or catamenial epilepsy. This seizure condition can be induced with pharmacologic agents, or by electrical stimulation in rodents, with suitable manipulation of neuroendocrine milieu (Reddy, 2009; Reddy and Ramanathan, 2012). This chapter describes currently available experimental models to study perimenstrual seizures and catamenial epilepsy for use in testing therapeutic strategies and molecular investigations of menstrual seizures. There are several features for an ideal catamenial epilepsy model. It should reflect a pathophysiology similar to those of catamenial seizures in women with epilepsy; exhibit appropriate menstrual seizure phenotype, consistent with the neuroendocrine fluctuations of women with epilepsy;
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FIGURE 62.1 (A,B) The neuroendocrine basis of seizure susceptibility during the menstrual cycle. (A) Neuroendocrine pathways of steroid hormone production. The synthesis and secretion of estrogens and progesterone (P) from the ovaries is controlled primarily by the hypothalamic GnRH and pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). Estrogens and P act on nuclear steroid receptors, while P-derived neurosteroids bind to membrane GABAA receptors. (B) Temporal relationship between ovarian hormones and occurrence of catamenial seizures. The upper panel illustrates the strong relationship between seizure frequency and estradiol/P levels. The lower panel illustrates the three types of catamenial epilepsy. The vertical [light gray bars (pink bars in the web version)] (left and right) represent the likely period for the perimenstrual (C1) type, while the vertical [light gray bars (pink bars in the web version)] (middle) represents the likely period for the periovulatory (C2) type. The horizontal dark gray bar (bottom) represents the inadequate luteal (C3) type with low level of allopregnanolone. (C,D) Mouse neurosteroid withdrawal (NSW) model of catamenial epilepsy. (C) Experimental protocol for the NSW model in mice. Fully kindled mice were treated with pregnant mare's serum gonadotropin (PMSG, 5 IU, s.c.) at 3:00 p.m., followed 48 h later human chorionic gonadotropin (HCG, 5 IU, s.c.) at 1:00 p.m. Then, on day 9, they were given finasteride (50 mg/kg, i.p.). (D) Plasma AP levels in mice following treatment with gonadotropins and finasteride (50 mg/kg, i.p.) for induction of NSW. The dramatic decline in neurosteroid levels 24 h after finasteride would create a state of NSW that may partly mimic the neuroendocrine milieu commonly observed around the perimenstrual period.
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exhibit appropriate latency, following steroid hormone fluctuations or withdrawal period; and respond to drug therapy with resistance to certain anticonvulsants. Because catamenial epilepsy is a complex neurological disorder that encompasses many causes and seizure phenotypes, it is highly unlikely that any single animal model will truly recapitulate the full spectrum of clinical catamenial seizure features. Therefore, it is necessary to screen potential therapeutic products, and investigate pathological mechanisms in a battery of animal models prior to clinical trials.
METHODS OF GENERATION (PROCEDURES) Recently, we developed two distinct mouse models of perimenstrual catamenial epilepsy (Gangisetty and Reddy, 2010; Reddy and Ramanathan, 2012). These models are based on the premise that seizure susceptibility decreases when neurosteroid levels are high (luteal phase), and increases during their withdrawal (perimenstrual periods) in females, in association with specific changes in the GABAA receptor subunit plasticity. First, we created a chronic seizure condition using the hippocampus kindling model in female mice. Second, the fully kindled mice were subjected to fluctuating levels of neurosteroids, mimicking the ovarian cycle. Here, we utilized two distinct pharmacological approaches to induce elevated neurosteroid levels: (1) chronic exogenous P treatment protocol, and (2) gonadotropin regimen for induction of endogenous synthesis. This chapter will focus on the gonadotropininduced neurosteroid synthesis and withdrawal paradigm, as it appears more physiologically relevant than the exogenous P treatment. In this model, elevated neurosteroid levels are induced by sequential gonadotropin treatment, and withdrawal induced by the neurosteroid synthesis inhibitor finasteride (Fig. 62.1C,D). This model is based on the premise that seizure susceptibility decreases when neurosteroid levels are high (luteal phase) and increases during their withdrawal (perimenstrual periods) in females, in association with specific changes in GABAA receptor subunit plasticity. We have found that the mouse model of perimenstrual catamenial epilepsy is useful for the investigation of disease mechanisms, and exploring the efficacy of new therapeutic approaches.
Animals Female adult C57BL6 mice, weighing 25–30 g, are used in the study. Mice were housed individually, with free access to food and water. The mice are housed in an environmentally controlled animal facility under a 12-h light/ dark cycle. As catamenial epilepsy only occurs in women of childbearing age, the mice used for this model should be
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cycling females. No differences between strains of mice are expected with this model.
Implantation of Electrode Mice are anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). A twisted bipolar stainless steel wire electrode (model MS303/1; Plastic One, Roanoke, VA) is stereotaxically implanted in the ventral right hippocampus (2.9 mm posterior, 3.0 mm lateral, and 3.0 mm below dura) (Franklin and Paxinos, 1997) and anchored with dental acrylic to three jeweler’s screws placed in the skull. A period of 7–10 days should be allowed for recovery.
Kindling Catamenial seizures are observed in women with preexisting epilepsy. Therefore, animal models should mimic this key criterion. Kindling provides a suitable background seizure model for developing a perimenstrual catamenial epilepsy model in female mice. Hippocampus kindling is a widely used model of human complex partial seizures. Unlike the pilocarpine-induced epilepsy model, the kindling model does not result in neuronal loss or spontaneous seizures (under normal physiological conditions), and maintains reproductive function in female animals. The stimulation paradigm consists of 1 ms duration, bipolar, square current pulses delivered at 60 Hz for 1 s by using a kindling stimulator (A-M Systems, Sequim, WA). The afterdischarge threshold (ADT) is determined by stimulation at 5-min intervals, beginning with an intensity of 25 µA, and increasing in increments of 25 µA, until an afterdischarge (AD) of at least 5 s is obtained. Stimulation on subsequent days used an intensity that was 125% of the threshold value. Seizure activity after each stimulation is rated according to the criterion of Racine (1972), as modified for the mouse: stage 0, no response or behavior arrest; stage 1, chewing or head nodding; stage 2, chewing and head nodding; stage 3, forelimb clonus; stage 4, bilateral forelimb clonus and rearing; and stage 5, falling. In our work, the AD was recorded from the hippocampus electrode with a Grass CP511 preamplifier (Astro-Med, West Warwick, RI), and stored in digital form by using Axoscope 8.1 (Molecular Devices, Sunnyvale, CA). AD duration is the total duration of hippocampus electrographic spike activity (amplitude >2 times baseline) occurring in a rhythmic pattern at a frequency >1 Hz. The day of AD threshold determination should be considered day 1 of kindling. Kindling stimulation was delivered daily until stage 5 seizures were elicited on three consecutive days. Mice were used for the neurosteroid withdrawal (NSW) paradigm when they consistently exhibited stage 5 seizures with stimulation, considered to be the “fully kindled” state.
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Gonadotropin-Induced Neurosteroid Withdrawal Paradigm A state of elevated neurosteroids is induced in female mice by a sequential gonadotropin regimen (Brooke et al., 2007). To produce prolonged elevated levels of P and neurosteroids that more closely model the luteal changes in women, mice are treated with pregnant mare’s serum gonadotropin (5 IU s.c.) at 3:00 p.m., followed 46 h later by human chorionic gonadotropin (5 IU s.c.) at 1:00 p.m. The day of the second gonadotropin injection is considered day 1 of elevated neurosteroids. On the morning of the ninth day, mice are injected with the 5α-reductase and neurosteroid synthesis inhibitor finasteride (50 mg/kg i.p.) to produce an abrupt decline in neurosteroid levels, to model more closely perimenstrual changes in women. Animals should be tested 24 h after finasteride administration (NSW). The control group receives saline injections. This protocol is similar to the standard P treatment approaches used previously for the induction of NSW (Moran and Smith, 1998; Smith et al., 1998a,b), and is also comparable with the pseudopregnancy model in rats (Reddy et al., 2001; Reddy and Rogawski, 2001). Although it is not practical to replicate the actual endocrine milieu of the menstrual cycle in mouse models, this endocrine state may be physiologically similar to the perimenstrual period. We did not use the gonadectomy model because of potential problems of interpretation associated with complete deficiency of ovarian-derived hormones; such animals need hormone replacements that may have variable effects on seizures, depending on the age, dose, and duration of treatment (Scharfman et al., 2005).
CHARACTERISTICS AND DEFINING FEATURES Endogenous Neurosteroids P plays a key role in catamenial epilepsy. P has consistent anticonvulsant and antiepileptic properties in animals and humans (Herzog, 1999; Jacono and Robinson, 1987; Reddy, 2009; Reddy and Jian, 2010). P has long been known to have antiseizure activity in a variety of animal models of epilepsy. In recent years, numerous studies have confirmed the powerful anticonvulsant activity of P in diverse animal seizure models (Reddy, 2004). Consequently, seizure susceptibility is very low during physiological conditions associated with high P. In women with epilepsy, natural cyclic variations in P during the menstrual cycle could influence catamenial seizure susceptibility (Fig. 62.1B). Seizures decrease in the mid-luteal phase, when serum P levels are high. Likewise, seizure activity increases premenstrually when P levels fall, and there is a decrease in the serum P-to-estrogen ratio. Changes in P levels have been directly correlated with catamenial seizures (Reddy, 2009).
The emerging evidence clearly indicates that perimenstrual catamenial seizures are associated with a rapid decline in P around menstruation. P is a precursor for the synthesis of neurosteroids in the brain. Neurosteroids are steroids that rapidly alter neuronal excitability through nongenomic mechanisms (Reddy, 2003). A variety of neurosteroids are known to be synthesized in the brain. The most widely studied are the P derivatives, allopregnanolone (AP) and pregnanolone. As neurosteroids are highly lipophilic, they readily cross the blood–brain barrier. It has been observed that neurosteroids synthesized in peripheral tissues accumulate in the brain (Do Rego et al., 2009; Reddy, 2010). Neurosteroids rapidly alter neuronal excitability through direct interaction with synaptic and extrasynaptic GABAA receptors (Fig. 62.1A). AP and other structurally related neurosteroids act as positive allosteric modulators and direct activators of GABAA receptors (Carver and Reddy, 2013). At low concentrations, neurosteroids potentiate GABAA receptor currents, whereas at higher concentrations, they directly activate the receptor. The GABAA receptor is a pentamer consisting of five subunits that form a chloride channel. Sixteen subunits (α1–6, β1–3, γ1–3, δ, ε, θ, and π subunits) have been identified so far. The effect of neurosteroids on GABAA receptors occurs by binding to discrete sites on the receptor–channel complex that are located within the transmembrane domains of the α and β subunits (Akk et al., 2005; Hosie et al., 2007; Chisari et al., 2010). The GABAA receptor mediates two types of GABAergic inhibition, now stratified into synaptic (phasic) or extrasynaptic (tonic) inhibition. Although neurosteroids act on all GABAA receptor isoforms, they have large effects on extrasynaptic δ-subunit GABAA receptors that mediate tonic currents (Carver and Reddy, 2013; Reddy and Rogawski, 2010; Wohlfarth et al., 2002). Thus, GABAA receptors that contain the δ subunit are highly sensitive to neurosteroid potentiation. Tonic currents cause a steady inhibition of neurons and reduce their excitability.
Genetic and Molecular Changes GABAA receptor subunit expression is not static in neurons, but undergoes alterations that compensate for changes in the endogenous hormonal milieu and to exogenously administered agents that modulate GABAA receptors, such as benzodiazepines or neurosteroids. It is now well recognized that prolonged exposure to AP in rats causes increased expression of the α4 receptor subunit in hippocampus, resulting in decreased benzodiazepine sensitivity of GABAA receptor currents (Gangisetty and Reddy, 2010; Smith et al., 1998a,b). Although α4 can assemble with γ2 to form synaptic GABAA receptors, it preferentially assembles with δ to form extrasynaptic GABAA receptors. Treatment with AP can result in transient increases in expression of the δ subunit in hippocampus, and increased
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benzodiazepine-insensitive tonic current. P also increases δ-subunit expression, likely as a result of its conversion to AP. The relevance of the increased δ-subunit expression for catamenial epilepsy is unclear, as δ-subunit increases may be transitory, and followed by reduced expression with chronic exposure, as in pregnancy, or in the prolonged luteal phase of the human menstrual cycle. To investigate the potential molecular mechanisms underlying the catamenial-like seizure exacerbations, we determined the changes in GABAA receptor subunit mRNA expression during NSW in the hippocampus, previously shown to exhibit neurosteroid-dependent plasticity (Gangisetty and Reddy, 2010; Maguire and Mody, 2008; Smith et al., 1998b). Twenty-four hours after NSW, the levels of α4 subunit were significantly increased, compared with its expression in the control group. The abundance of α-subunit mRNA in the hippocampus was also significantly increased in withdrawn animals. In contrast, no changes in levels of β2- and γ2-subunit expression were observed 24 h after NSW. Overall, these findings indicate a marked increase in the expression of α4 and δ subunits in the hippocampus during NSW in the mouse catamenial paradigm. Although the dentate gyrus normally has high expression of α4 and α subunits, they are also expressed in CA1 hippocampus, in response to fluctuations in neurosteroids (Smith et al., 1998a).
MONITORING Seizure Susceptibility in Fully Kindled Mice To determine whether NSW is associated with heightened seizure susceptibility, we analyzed the stimulation-evoked seizure activity in animals undergoing NSW. Fully kindled mice were subjected to the NSW protocol as described previously. Four parameters were assessed as indices of seizure propensity: (1) ADT current for generalized seizures, (2) stimulation-induced electrographic ADT duration, (3) behavioral seizure intensity measured as per the Racine scale, and (4) duration of generalized seizures. Consistent with heightened excitability, there was a marked decrease in the ADT current to induce generalized seizures at 24 h after NSW (mean ADT value, 105 and 60 µA for control and withdrawal, respectively). The mean duration of the individual generalized seizures was longer in withdrawal than in control animals. The total duration of AD was significantly higher in withdrawal animals. The number of animals exhibiting generalized seizures at 50% ADT current was significantly higher after NSW than in the control group. This response was significantly higher 12 and 24 h after NSW, and returned to control level by 48 h after withdrawal, indicating a transitory period for seizure exacerbation after NSW. Neurosteroid-withdrawn animals showed continuous bursts of spikes that progressively increased in
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amplitude and duration, indicating heightened epileptiform activity. The electrographic AD duration was increased markedly 12 h after withdrawal, reached a maximal level 24 h after withdrawal, and declined to almost control level by 48 h after withdrawal. Finasteride did not cause such seizure exacerbations in fully kindled control (nonwithdrawing) animals, indicating the specificity of NSW on the exacerbation of seizure activity in fully kindled mice.
Spontaneous Perimenstrual Seizures NSW in a few fully kindled mice (∼15%) triggered the appearance of spontaneous perimenstrual seizures. In this study, spontaneous perimenstrual seizures were defined as those observed prior to kindling, or at least 4 min after the kindling stimulation at their regular ADT, and typically after stage 3/4 seizures. Spontaneous seizures in our study lasted transiently within 2–8 h following NSW induction. Spontaneous seizures resembling stage 6 seizures (wild running, jumping, and vocalizations) are evident, but there were limited long-term monitoring studies to determine their frequency or severity.
Persistence of Seizure Exacerbation Kindling seizures are permanent and, similarly, the NSWinduced exacerbation of kindled seizures persisted for four cycles, or longer. For instance, mice that recovered from an NSW cycle have been subjected to another cycle. Stimulation-elicited perimenstrual-like seizures are consistently observed in up to four cycles, with an intercycle interval of 2 weeks.
Neuropathology and Neuroimaging Histological examination of NSW mice has not shown any significant differences, when compared to control mice of the same age and sex.
Species Variations Based on the NSW approach, a rat model of perimenstrual catamenial epilepsy has also been developed (Reddy et al., 2001). Rodents have a 4- to 5-day estrous cycle, and studies of fluctuations in seizure susceptibility in cycling female rats have not led to results that are relevant to the human menstrual cycle. To provide a model that more closely mimics the human situation, a condition of elevated P was created in rats by gonadotropin treatment. This resulted in prolonged high circulating levels of P, similar to those that occur in the luteal phase of the menstrual cycle. Then, to simulate the withdrawal of AP that occurs at the time of menstruation, the animals were treated with finasteride 11 days after the initiation of gonadotropin treatment. Withdrawal
938 PART | IX Modeling Conditions that Predispose to Seizures or Epilepsy
of neurosteroids had led to decreased seizure threshold, and increased seizure activity (Reddy et al., 2001). This paradigm was also verified in female epileptic rats with spontaneous seizures (Lawrence et al., 2010; Reddy and Zeng, 2007).
RESPONSE TO ANTIEPILEPTIC DRUGS Diagnostic Testing or Validation of Model To examine the ability of test drugs to suppress the expression of kindled seizures, fully kindled animals were tested 24 h after the induction of NSW. Two different drugs, diazepam and AP, were selected for pharmacological evaluation in neurosteroid-withdrawn animals. Diazepam is a benzodiazepine-site agonist at α1-, α2-, or α3-containing GABAA receptors with potent antiseizure activity, but it is insensitive as an agonist at α4-containing GABAA receptors expressed in the hippocampus (Gulinello et al., 2001; Smith et al., 1998b). The neurosteroid AP binds to all isoforms, but has enhanced sensitivity at the δ-subunit containing GABAA receptors. In the kindling studies, animals were tested for drug sensitivity 24 h after NSW. On the day of testing, animals were injected intraperitoneally with diazepam (0.1–1 mg/kg) or AP (1–10 mg/kg), 15 min before kindling stimulations. Control animals were injected similarly with vehicle (15% cyclodextrin). During each stimulation session, the behavioral seizure score and the AD duration were noted. Diazepam produced a dose-dependent suppression of behavioral seizure activity, and AD duration with significant effects at 0.1, 0.3, and 1 mg/kg in control (nonwithdrawal) animals, confirming diazepam protection against hippocampus kindling-induced seizures. In contrast, mice undergoing NSW had significantly decreased seizure protection by diazepam. At a dose of 1 mg/kg, diazepam produced an average of 95% and 30% decrease in seizure expression, in control and withdrawal groups, respectively. Taken together, these results are consistent with the notion that NSW causes relative insensitivity to diazepam caused by increased α4-containing GABAA receptor expression in the hippocampus. In addition, we investigated the efficacy of the prototype neurosteroid AP in control and NSW mice 24 h after NSW. Fully kindled control and neurosteroid-withdrawn mice were tested in the hippocampal kindling model with three doses of AP (1, 5, and 10 mg/kg s.c.). At these doses, AP exerted dose-dependent suppression of the behavioral seizures, and AD duration, in both control and NSW mice. Interestingly, NSW mice exhibited enhanced neurosteroid sensitivity, and greater AP-induced suppression of seizure severity, and AD duration, than in control mice. Moreover, plasma levels of AP achieved at various doses of AP treatment were similar between control and withdrawn groups,
especially without significant drug accumulation in withdrawn animals, indicating that AP sensitivity was not caused by pharmacokinetic factors (Reddy and Ramanathan, 2012). The synthetic neurosteroid ganaxolone (1, 3, and 10 mg/kg) also produced enhanced (60%) efficacy in fully kindled neurosteroid-withdrawn animals, confirming the enhanced sensitivity to neurosteroids in the NSW model of catamenial epilepsy (Reddy and Rogawski, 2001).
LIMITATIONS The validation of animal models of catamenial epilepsy requires certain criteria to be met for them to be representative of the human condition. They should have a close similarity in eliciting an epilepsy-like state, as well as a pathophysiology that mirrors the disease in humans. The NSW model partly meets these criteria and certainly offers some advantages over the use of conventional seizure models (Reddy, 2009). These models are based on similar physiological dynamics of ovarian P secretion during the menstrual cycle that cannot be simulated in the exogenous drug delivery models. Moreover, the 9-day elevation of P and AP levels in the rodent model closely matches the 10-day increase in AP levels during the luteal phase of the menstrual cycle. Although the exact etiology of catamenial epilepsy is not completely understood, the NSW model better simulates changes in the AP-to-estrogen ratio that is believed to be critical for perimenstrual catamenial epilepsy. However, the actual endocrine conditions that exist in the menstrual cycle are different from those observed in animal models of catamenial epilepsy. This is a major concern with most animal models developed in rodents. In rodents, the estrous cycle duration is 4–7 days, and the menstrual cycle in women lasts about 28 days.
INSIGHTS ON NEUROSTEROID WITHDRAWAL: WHAT THIS PARADIGM IS GOOD FOR Catamenial epilepsy affects a high proportion of women of reproductive age with drug refractory epilepsy. It is a multifaceted neuroendocrine condition (El-Khayat et al., 2008; Tuveri et al., 2008). Neurosteroids play a key role in the pathophysiology of catamenial epilepsy. Perimenstrual catamenial epilepsy is believed to be due to the withdrawal of neurosteroids as a result of the fall in P at the time of menstruation. There are currently no specific treatments for catamenial seizures. The NSW model of catamenial epilepsy has been used to investigate therapies for catamenial epilepsy. A key result is the observation that conventional antiepileptic drugs have reduced potency in protecting against catamenial seizures. This finding is consistent with women with catamenial epilepsy who do not respond to these drugs. The model therefore represents a
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form of the pharmacoresistance paradigm. The increased relative expression of benzodiazepine-insensitive GABAA receptors at the time of NSW likely accounts for the reduced activity of benzodiazepines. We unexpectedly found that neurosteroids, including their synthetic analogs, had enhanced activity in the perimenstrual catamenial epilepsy model (Reddy and Rogawski, 2000a,b, 2001). Based on our studies in catamenial epilepsy models, we suggested that “neurosteroid replacement” may be a useful approach to prevent catamenial seizure exacerbations (Reddy and Rogawski, 2009). A neurosteroid or a neurosteroid-like compound, such as ganaxolone, could be administered in a “pulse” prior to menstruation, and then withdrawn, or continuously administered throughout the month at low doses to avoid sedative side effects. Such low doses are expected to contribute little anticonvulsant activity during most of the menstrual cycle. Therefore, neurosteroids might provide an effective approach for catamenial epilepsy therapy. The molecular mechanisms underlying such enhanced neurosteroid sensitivity in catamenial epilepsy remain unclear. A δ-force hypothesis was coined to explain this phenomenon (Galanopoulou, 2015; Reddy, 2014). Recently, we found a novel plasticity of extrasynaptic δ-containing GABAA receptors in the dentate gyrus in a mouse perimenstrual model (Carver et al., 2014; Wu et al., 2013). In summary, the proposed mechanism poses that the perimenstrual decline in neurosteroids triggers the selective overexpression of α4δ subunits in the dentate gyrus granule cells, but this is not sufficient to suppress the increased seizure susceptibility of NSW mice in the absence of AP. Yet, this pathologic increase in α4δ subunits may act as a vehiclecatalyst (Trojan horse) for the exogenous AP to inhibit seizures. Indeed, this special sensitivity to AP that woman with perimenstrual epilepsy exhibit is in agreement with the findings from the NIH P trial, in which the responder group was women with the C1 type of catamenial epilepsy (Herzog et al., 2012). Interestingly, these women responders demonstrated significant posttreatment increase in AP levels (Herzog et al., 2014). This scenario would render perimenstrual epilepsy an ideal candidate to test the therapeutic effects of neurosteroids as a brief pulse treatment protocol during this perimenstrual period. Therefore, these findings may represent a molecular rationale for neurosteroid therapy of catamenial epilepsy.
ACKNOWLEDGMENT The author thanks Bryan Clossen and Victoria Golub for reading the manuscript.
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Lawrence, C., Martin, B.S., Sun, C., Williamson, J., Kapur, J., 2010. Endogenous neurosteroid synthesis modulates seizure frequency. Ann. Neurol. 67, 689–693. Maguire, J., Mody, I., 2008. GABAAR plasticity during pregnancy: relevance to postpartum depression. Neuron 59, 207–213. Moran, M.H., Smith, S.S., 1998. Progesterone withdrawal I: pro-convulsant effects. Brain Res. 807, 84–90. Newmark, M.E., Penry, J.K., 1980. Catamenial epilepsy: a review. Epilepsia 21, 281–300. Quigg, M., Smithson, S.D., Fowler, K.M., Sursal, T., Herzog, A.G., 2009. NIH Progesterone Trial Study Group: laterality and location influence catamenial seizure expression in women with partial epilepsy. Neurology 73 (3), 223–227. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr. Clin. Neurophysiol. 32, 281–294. Reddy, D.S., 2003. Pharmacology of endogenous neuroactive steroids. Crit. Rev. Neurobiol. 15, 197–234. Reddy, D.S., 2004. Testosterone modulation of seizure susceptibility is mediated by neurosteroids 17β-estradiol and 3α-androstanediol. Neuroscience 129, 195–207. Reddy, D.S., 2009. The role of neurosteroids in the pathophysiology and treatment of catamenial epilepsy. Epilepsy Res. 85, 1–30. Reddy, D.S., 2010. Neurosteroids: endogenous role in the human brain and therapeutic potentials. Prog. Brain Res. 186, 113–137. Reddy, D.S., 2014. Neurosteroids and their role in sex-specific epilepsies. Neurobiol. Dis. 72B, 198–209. Reddy, D.D., Jian, K., 2010. The testosterone-derived neurosteroid androstanediol is a positive allosteric modulator of GABAA receptors. J. Pharmacol. Exp. Ther. 334, 1031–1041. Reddy, D.S., Ramanathan, G., 2012. Finasteride inhibits the disease-modifying activity of progesterone in the hippocampus kindling model of epileptogenesis. Epilepsy Behav. 25, 92–97. Reddy, D.S., Rogawski, M.A., 2000a. Chronic treatment with the neuroactive steroid ganaxolone in the rat induces anticonvulsant tolerance to diazepam but not to itself. J. Pharmacol. Exp. Ther. 295, 1241–1248. Reddy, D.S., Rogawski, M.A., 2000b. Enhanced anticonvulsant activity of ganaxolone after neurosteroid withdrawal in a rat model of catamenial epilepsy. J. Pharmacol. Exp. Ther. 294, 909–915.
Reddy, D.S., Rogawski, M.A., 2001. Enhanced anticonvulsant activity of neuroactive steroids in a rat model of catamenial epilepsy. Epilepsia 42, 337–344. Reddy, D.S., Rogawski, M.A., 2009. Neurosteroid replacement therapy for catamenial epilepsy. Neurotherapy 6 (2), 392–401. Reddy, D.S., Rogawski, M.A., 2010. Ganaxolone suppression of behavioral and electrographic seizures in the mouse amygdala kindling model. Epilepsy Res. 89, 254–260. Reddy, D.S., Zeng, Y.C., 2007. Effect of neurosteroid withdrawal on spontaneous recurrent seizures in a rat model of catamenial epilepsy. FASEB J. 21, 885.14. Reddy, D.S., Kim, H.Y., Rogawski, M.A., 2001. Neurosteroid withdrawal model of perimenstrual catamenial epilepsy. Epilepsia 42, 328–336. Scharfman, H.E., Goodman, J.H., Rigoulot, M.A., Berger, R.E., Walling, S.G., Mercurio, T.C., Stormes, K., Maclusky, N.J., 2005. Seizure susceptibility in intact and ovariectomized female rats treated with the convulsant pilocarpine. Exp. Neurol. 196, 73–86. Smith, S.S., Gong, Q.H., Hsu, F.C., Markowitz, R.S., ffrench-Mullen, J.M., Li, X., 1998a. GABAA receptor α4 subunit suppression prevents withdrawal properties of an endogenous steroid. Nature 392, 926–930. Smith, S.S., Gong, Q.H., Li, X., Moran, M.H., Bitran, D., Frye, C.A., Hsu, F.C., 1998b. Withdrawal from 3α-OH-5α-pregnan-20-One using a pseudopregnancy model alters the kinetics of hippocampal GABAAgated current and increases the GABAA receptor α4-subunit in association with increased anxiety. J. Neurosci. 18, 5275–5284. Tuveri, A., Paoletti, A.M., Orrù, M., Melis, G.B., Marotto, M.F., Zedda, P., Marrosu, F., Sogliano, C., Marra, C., Biggio, G., Concas, A, 2008. Reduced serum level of THDOC, an anticonvulsant steroid, in women with perimenstrual catamenial epilepsy. Epilepsia 49, 1221–1229. Wohlfarth, K.M., Bianchi, M.T., Macdonald, R.L., 2002. Enhanced neurosteroid potentiation of ternary GABAA receptors containing the δ subunit. J. Neurosci. 22, 1541–1549. Wu, X., Gangisetty, O., Carver, C.M., Reddy, D.S., 2013. Estrous cycle regulation of extrasynaptic δ-containing GABAA receptor-mediated tonic inhibition and limbic epileptogenesis. J. Pharmacol. Exp. Ther. 346, 146–160.
Perimenstrual Seizures and Neurosteroid Withdrawal Doodipala S. Reddy Texas A&M Health Science Center, Bryan, TX, United States
GENERAL DESCRIPTION The term “catamenial epilepsy” is used to describe the cyclical occurrence of seizure exacerbations during particular phases of the menstrual cycle in women with preexisting epilepsy (Herzog et al., 2008; Newmark and Penry, 1980; Reddy, 2004). The types of epilepsies and seizures that are susceptible to catamenial fluctuations are not yet thoroughly defined. However, it appears that seizures in both partial epilepsies (such as mesial temporal lobe epilepsy) and certain primary generalized epilepsies (such as juvenile myoclonic epilepsy) can exhibit catamenial exacerbations. Of note, menstrual cycle influence on seizure susceptibility has been anecdotally observed since antiquity. However, because women with epilepsies that are severe enough to exhibit cyclical changes in seizure frequency are invariably treated with antiepileptic drugs, catamenial epilepsy is now defined as a specific form of pharmacoresistant epilepsy. With standard antiepileptic drug treatment, some of these women experience a resolution of seizures except at certain times during the menstrual cycle, while others do not respond to medications at all, or display inconsistent therapeutic benefit. In either case, the subjects continue to have intractable seizures, despite the best efforts of modern medicine. Catamenial seizure exacerbations affect between 25% and 70% of women with epilepsy who are of reproductive age (Reddy, 2014). The reason for the large range is due to differences in definition; however, three types of catamenial seizures have been identified: perimenstrual (C1), periovulatory (C2), and inadequate luteal phase (C3), based on when a patient observes increases in seizure frequency/ severity (Herzog et al., 1997; Reddy, 2009) (Fig. 62.1A,B). The perimenstrual is the most common clinical type. The specific pattern of incidence can be identified simply by charting menses and seizure clusters; along with obtaining
Models of Seizures and Epilepsy. http://dx.doi.org/10.1016/B978-0-12-804066-9.00063-8 Copyright © 2017 Elsevier Inc. All rights reserved.
mid-luteal phase serum progesterone (P) levels to distinguish between normal and inadequate luteal phase cycles (Herzog et al., 2008; Quigg et al., 2009). The diagnosis of catamenial epilepsy is mainly based on the assessment of menstruation and seizure records. Using the first day of menstrual bleeding as the first day of the cycle, the menstrual cycle is divided into four phases: (1) menstrual phase, days –3 to +3; (2) follicular phase, days +4 to +9; (3) ovulatory phase, days +10 to +16; and (4) luteal phase, days +17 to –4. The number of seizures in each phase is counted for at least two cycles, and a twofold or greater increase in frequency during a particular phase of the menstrual cycle can be used as diagnostic criteria of catamenial epilepsy. In perimenstrual catamenial epilepsy (C1), women with epilepsy experience an increase in seizure activity before, during, or after the onset of menstruation (Reddy, 2009). Catamenial epilepsy is observed in women with both ovulatory and anovulatory cycles. In one study (Herzog et al., 2004), about 16.5% of subjects were found to have anovulatory cycles. These women showed a third type of catamenial epilepsy, referred to as inadequate luteal phase (C3) or anovulatory luteal seizures. Experimental models have been developed that mimic the perimenstrual seizures or catamenial epilepsy. This seizure condition can be induced with pharmacologic agents, or by electrical stimulation in rodents, with suitable manipulation of neuroendocrine milieu (Reddy, 2009; Reddy and Ramanathan, 2012). This chapter describes currently available experimental models to study perimenstrual seizures and catamenial epilepsy for use in testing therapeutic strategies and molecular investigations of menstrual seizures. There are several features for an ideal catamenial epilepsy model. It should reflect a pathophysiology similar to those of catamenial seizures in women with epilepsy; exhibit appropriate menstrual seizure phenotype, consistent with the neuroendocrine fluctuations of women with epilepsy;
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FIGURE 62.1 (A,B) The neuroendocrine basis of seizure susceptibility during the menstrual cycle. (A) Neuroendocrine pathways of steroid hormone production. The synthesis and secretion of estrogens and progesterone (P) from the ovaries is controlled primarily by the hypothalamic GnRH and pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). Estrogens and P act on nuclear steroid receptors, while P-derived neurosteroids bind to membrane GABAA receptors. (B) Temporal relationship between ovarian hormones and occurrence of catamenial seizures. The upper panel illustrates the strong relationship between seizure frequency and estradiol/P levels. The lower panel illustrates the three types of catamenial epilepsy. The vertical [light gray bars (pink bars in the web version)] (left and right) represent the likely period for the perimenstrual (C1) type, while the vertical [light gray bars (pink bars in the web version)] (middle) represents the likely period for the periovulatory (C2) type. The horizontal dark gray bar (bottom) represents the inadequate luteal (C3) type with low level of allopregnanolone. (C,D) Mouse neurosteroid withdrawal (NSW) model of catamenial epilepsy. (C) Experimental protocol for the NSW model in mice. Fully kindled mice were treated with pregnant mare's serum gonadotropin (PMSG, 5 IU, s.c.) at 3:00 p.m., followed 48 h later human chorionic gonadotropin (HCG, 5 IU, s.c.) at 1:00 p.m. Then, on day 9, they were given finasteride (50 mg/kg, i.p.). (D) Plasma AP levels in mice following treatment with gonadotropins and finasteride (50 mg/kg, i.p.) for induction of NSW. The dramatic decline in neurosteroid levels 24 h after finasteride would create a state of NSW that may partly mimic the neuroendocrine milieu commonly observed around the perimenstrual period.
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exhibit appropriate latency, following steroid hormone fluctuations or withdrawal period; and respond to drug therapy with resistance to certain anticonvulsants. Because catamenial epilepsy is a complex neurological disorder that encompasses many causes and seizure phenotypes, it is highly unlikely that any single animal model will truly recapitulate the full spectrum of clinical catamenial seizure features. Therefore, it is necessary to screen potential therapeutic products, and investigate pathological mechanisms in a battery of animal models prior to clinical trials.
METHODS OF GENERATION (PROCEDURES) Recently, we developed two distinct mouse models of perimenstrual catamenial epilepsy (Gangisetty and Reddy, 2010; Reddy and Ramanathan, 2012). These models are based on the premise that seizure susceptibility decreases when neurosteroid levels are high (luteal phase), and increases during their withdrawal (perimenstrual periods) in females, in association with specific changes in the GABAA receptor subunit plasticity. First, we created a chronic seizure condition using the hippocampus kindling model in female mice. Second, the fully kindled mice were subjected to fluctuating levels of neurosteroids, mimicking the ovarian cycle. Here, we utilized two distinct pharmacological approaches to induce elevated neurosteroid levels: (1) chronic exogenous P treatment protocol, and (2) gonadotropin regimen for induction of endogenous synthesis. This chapter will focus on the gonadotropininduced neurosteroid synthesis and withdrawal paradigm, as it appears more physiologically relevant than the exogenous P treatment. In this model, elevated neurosteroid levels are induced by sequential gonadotropin treatment, and withdrawal induced by the neurosteroid synthesis inhibitor finasteride (Fig. 62.1C,D). This model is based on the premise that seizure susceptibility decreases when neurosteroid levels are high (luteal phase) and increases during their withdrawal (perimenstrual periods) in females, in association with specific changes in GABAA receptor subunit plasticity. We have found that the mouse model of perimenstrual catamenial epilepsy is useful for the investigation of disease mechanisms, and exploring the efficacy of new therapeutic approaches.
Animals Female adult C57BL6 mice, weighing 25–30 g, are used in the study. Mice were housed individually, with free access to food and water. The mice are housed in an environmentally controlled animal facility under a 12-h light/ dark cycle. As catamenial epilepsy only occurs in women of childbearing age, the mice used for this model should be
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cycling females. No differences between strains of mice are expected with this model.
Implantation of Electrode Mice are anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). A twisted bipolar stainless steel wire electrode (model MS303/1; Plastic One, Roanoke, VA) is stereotaxically implanted in the ventral right hippocampus (2.9 mm posterior, 3.0 mm lateral, and 3.0 mm below dura) (Franklin and Paxinos, 1997) and anchored with dental acrylic to three jeweler’s screws placed in the skull. A period of 7–10 days should be allowed for recovery.
Kindling Catamenial seizures are observed in women with preexisting epilepsy. Therefore, animal models should mimic this key criterion. Kindling provides a suitable background seizure model for developing a perimenstrual catamenial epilepsy model in female mice. Hippocampus kindling is a widely used model of human complex partial seizures. Unlike the pilocarpine-induced epilepsy model, the kindling model does not result in neuronal loss or spontaneous seizures (under normal physiological conditions), and maintains reproductive function in female animals. The stimulation paradigm consists of 1 ms duration, bipolar, square current pulses delivered at 60 Hz for 1 s by using a kindling stimulator (A-M Systems, Sequim, WA). The afterdischarge threshold (ADT) is determined by stimulation at 5-min intervals, beginning with an intensity of 25 µA, and increasing in increments of 25 µA, until an afterdischarge (AD) of at least 5 s is obtained. Stimulation on subsequent days used an intensity that was 125% of the threshold value. Seizure activity after each stimulation is rated according to the criterion of Racine (1972), as modified for the mouse: stage 0, no response or behavior arrest; stage 1, chewing or head nodding; stage 2, chewing and head nodding; stage 3, forelimb clonus; stage 4, bilateral forelimb clonus and rearing; and stage 5, falling. In our work, the AD was recorded from the hippocampus electrode with a Grass CP511 preamplifier (Astro-Med, West Warwick, RI), and stored in digital form by using Axoscope 8.1 (Molecular Devices, Sunnyvale, CA). AD duration is the total duration of hippocampus electrographic spike activity (amplitude >2 times baseline) occurring in a rhythmic pattern at a frequency >1 Hz. The day of AD threshold determination should be considered day 1 of kindling. Kindling stimulation was delivered daily until stage 5 seizures were elicited on three consecutive days. Mice were used for the neurosteroid withdrawal (NSW) paradigm when they consistently exhibited stage 5 seizures with stimulation, considered to be the “fully kindled” state.
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Gonadotropin-Induced Neurosteroid Withdrawal Paradigm A state of elevated neurosteroids is induced in female mice by a sequential gonadotropin regimen (Brooke et al., 2007). To produce prolonged elevated levels of P and neurosteroids that more closely model the luteal changes in women, mice are treated with pregnant mare’s serum gonadotropin (5 IU s.c.) at 3:00 p.m., followed 46 h later by human chorionic gonadotropin (5 IU s.c.) at 1:00 p.m. The day of the second gonadotropin injection is considered day 1 of elevated neurosteroids. On the morning of the ninth day, mice are injected with the 5α-reductase and neurosteroid synthesis inhibitor finasteride (50 mg/kg i.p.) to produce an abrupt decline in neurosteroid levels, to model more closely perimenstrual changes in women. Animals should be tested 24 h after finasteride administration (NSW). The control group receives saline injections. This protocol is similar to the standard P treatment approaches used previously for the induction of NSW (Moran and Smith, 1998; Smith et al., 1998a,b), and is also comparable with the pseudopregnancy model in rats (Reddy et al., 2001; Reddy and Rogawski, 2001). Although it is not practical to replicate the actual endocrine milieu of the menstrual cycle in mouse models, this endocrine state may be physiologically similar to the perimenstrual period. We did not use the gonadectomy model because of potential problems of interpretation associated with complete deficiency of ovarian-derived hormones; such animals need hormone replacements that may have variable effects on seizures, depending on the age, dose, and duration of treatment (Scharfman et al., 2005).
CHARACTERISTICS AND DEFINING FEATURES Endogenous Neurosteroids P plays a key role in catamenial epilepsy. P has consistent anticonvulsant and antiepileptic properties in animals and humans (Herzog, 1999; Jacono and Robinson, 1987; Reddy, 2009; Reddy and Jian, 2010). P has long been known to have antiseizure activity in a variety of animal models of epilepsy. In recent years, numerous studies have confirmed the powerful anticonvulsant activity of P in diverse animal seizure models (Reddy, 2004). Consequently, seizure susceptibility is very low during physiological conditions associated with high P. In women with epilepsy, natural cyclic variations in P during the menstrual cycle could influence catamenial seizure susceptibility (Fig. 62.1B). Seizures decrease in the mid-luteal phase, when serum P levels are high. Likewise, seizure activity increases premenstrually when P levels fall, and there is a decrease in the serum P-to-estrogen ratio. Changes in P levels have been directly correlated with catamenial seizures (Reddy, 2009).
The emerging evidence clearly indicates that perimenstrual catamenial seizures are associated with a rapid decline in P around menstruation. P is a precursor for the synthesis of neurosteroids in the brain. Neurosteroids are steroids that rapidly alter neuronal excitability through nongenomic mechanisms (Reddy, 2003). A variety of neurosteroids are known to be synthesized in the brain. The most widely studied are the P derivatives, allopregnanolone (AP) and pregnanolone. As neurosteroids are highly lipophilic, they readily cross the blood–brain barrier. It has been observed that neurosteroids synthesized in peripheral tissues accumulate in the brain (Do Rego et al., 2009; Reddy, 2010). Neurosteroids rapidly alter neuronal excitability through direct interaction with synaptic and extrasynaptic GABAA receptors (Fig. 62.1A). AP and other structurally related neurosteroids act as positive allosteric modulators and direct activators of GABAA receptors (Carver and Reddy, 2013). At low concentrations, neurosteroids potentiate GABAA receptor currents, whereas at higher concentrations, they directly activate the receptor. The GABAA receptor is a pentamer consisting of five subunits that form a chloride channel. Sixteen subunits (α1–6, β1–3, γ1–3, δ, ε, θ, and π subunits) have been identified so far. The effect of neurosteroids on GABAA receptors occurs by binding to discrete sites on the receptor–channel complex that are located within the transmembrane domains of the α and β subunits (Akk et al., 2005; Hosie et al., 2007; Chisari et al., 2010). The GABAA receptor mediates two types of GABAergic inhibition, now stratified into synaptic (phasic) or extrasynaptic (tonic) inhibition. Although neurosteroids act on all GABAA receptor isoforms, they have large effects on extrasynaptic δ-subunit GABAA receptors that mediate tonic currents (Carver and Reddy, 2013; Reddy and Rogawski, 2010; Wohlfarth et al., 2002). Thus, GABAA receptors that contain the δ subunit are highly sensitive to neurosteroid potentiation. Tonic currents cause a steady inhibition of neurons and reduce their excitability.
Genetic and Molecular Changes GABAA receptor subunit expression is not static in neurons, but undergoes alterations that compensate for changes in the endogenous hormonal milieu and to exogenously administered agents that modulate GABAA receptors, such as benzodiazepines or neurosteroids. It is now well recognized that prolonged exposure to AP in rats causes increased expression of the α4 receptor subunit in hippocampus, resulting in decreased benzodiazepine sensitivity of GABAA receptor currents (Gangisetty and Reddy, 2010; Smith et al., 1998a,b). Although α4 can assemble with γ2 to form synaptic GABAA receptors, it preferentially assembles with δ to form extrasynaptic GABAA receptors. Treatment with AP can result in transient increases in expression of the δ subunit in hippocampus, and increased
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benzodiazepine-insensitive tonic current. P also increases δ-subunit expression, likely as a result of its conversion to AP. The relevance of the increased δ-subunit expression for catamenial epilepsy is unclear, as δ-subunit increases may be transitory, and followed by reduced expression with chronic exposure, as in pregnancy, or in the prolonged luteal phase of the human menstrual cycle. To investigate the potential molecular mechanisms underlying the catamenial-like seizure exacerbations, we determined the changes in GABAA receptor subunit mRNA expression during NSW in the hippocampus, previously shown to exhibit neurosteroid-dependent plasticity (Gangisetty and Reddy, 2010; Maguire and Mody, 2008; Smith et al., 1998b). Twenty-four hours after NSW, the levels of α4 subunit were significantly increased, compared with its expression in the control group. The abundance of α-subunit mRNA in the hippocampus was also significantly increased in withdrawn animals. In contrast, no changes in levels of β2- and γ2-subunit expression were observed 24 h after NSW. Overall, these findings indicate a marked increase in the expression of α4 and δ subunits in the hippocampus during NSW in the mouse catamenial paradigm. Although the dentate gyrus normally has high expression of α4 and α subunits, they are also expressed in CA1 hippocampus, in response to fluctuations in neurosteroids (Smith et al., 1998a).
MONITORING Seizure Susceptibility in Fully Kindled Mice To determine whether NSW is associated with heightened seizure susceptibility, we analyzed the stimulation-evoked seizure activity in animals undergoing NSW. Fully kindled mice were subjected to the NSW protocol as described previously. Four parameters were assessed as indices of seizure propensity: (1) ADT current for generalized seizures, (2) stimulation-induced electrographic ADT duration, (3) behavioral seizure intensity measured as per the Racine scale, and (4) duration of generalized seizures. Consistent with heightened excitability, there was a marked decrease in the ADT current to induce generalized seizures at 24 h after NSW (mean ADT value, 105 and 60 µA for control and withdrawal, respectively). The mean duration of the individual generalized seizures was longer in withdrawal than in control animals. The total duration of AD was significantly higher in withdrawal animals. The number of animals exhibiting generalized seizures at 50% ADT current was significantly higher after NSW than in the control group. This response was significantly higher 12 and 24 h after NSW, and returned to control level by 48 h after withdrawal, indicating a transitory period for seizure exacerbation after NSW. Neurosteroid-withdrawn animals showed continuous bursts of spikes that progressively increased in
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amplitude and duration, indicating heightened epileptiform activity. The electrographic AD duration was increased markedly 12 h after withdrawal, reached a maximal level 24 h after withdrawal, and declined to almost control level by 48 h after withdrawal. Finasteride did not cause such seizure exacerbations in fully kindled control (nonwithdrawing) animals, indicating the specificity of NSW on the exacerbation of seizure activity in fully kindled mice.
Spontaneous Perimenstrual Seizures NSW in a few fully kindled mice (∼15%) triggered the appearance of spontaneous perimenstrual seizures. In this study, spontaneous perimenstrual seizures were defined as those observed prior to kindling, or at least 4 min after the kindling stimulation at their regular ADT, and typically after stage 3/4 seizures. Spontaneous seizures in our study lasted transiently within 2–8 h following NSW induction. Spontaneous seizures resembling stage 6 seizures (wild running, jumping, and vocalizations) are evident, but there were limited long-term monitoring studies to determine their frequency or severity.
Persistence of Seizure Exacerbation Kindling seizures are permanent and, similarly, the NSWinduced exacerbation of kindled seizures persisted for four cycles, or longer. For instance, mice that recovered from an NSW cycle have been subjected to another cycle. Stimulation-elicited perimenstrual-like seizures are consistently observed in up to four cycles, with an intercycle interval of 2 weeks.
Neuropathology and Neuroimaging Histological examination of NSW mice has not shown any significant differences, when compared to control mice of the same age and sex.
Species Variations Based on the NSW approach, a rat model of perimenstrual catamenial epilepsy has also been developed (Reddy et al., 2001). Rodents have a 4- to 5-day estrous cycle, and studies of fluctuations in seizure susceptibility in cycling female rats have not led to results that are relevant to the human menstrual cycle. To provide a model that more closely mimics the human situation, a condition of elevated P was created in rats by gonadotropin treatment. This resulted in prolonged high circulating levels of P, similar to those that occur in the luteal phase of the menstrual cycle. Then, to simulate the withdrawal of AP that occurs at the time of menstruation, the animals were treated with finasteride 11 days after the initiation of gonadotropin treatment. Withdrawal
938 PART | IX Modeling Conditions that Predispose to Seizures or Epilepsy
of neurosteroids had led to decreased seizure threshold, and increased seizure activity (Reddy et al., 2001). This paradigm was also verified in female epileptic rats with spontaneous seizures (Lawrence et al., 2010; Reddy and Zeng, 2007).
RESPONSE TO ANTIEPILEPTIC DRUGS Diagnostic Testing or Validation of Model To examine the ability of test drugs to suppress the expression of kindled seizures, fully kindled animals were tested 24 h after the induction of NSW. Two different drugs, diazepam and AP, were selected for pharmacological evaluation in neurosteroid-withdrawn animals. Diazepam is a benzodiazepine-site agonist at α1-, α2-, or α3-containing GABAA receptors with potent antiseizure activity, but it is insensitive as an agonist at α4-containing GABAA receptors expressed in the hippocampus (Gulinello et al., 2001; Smith et al., 1998b). The neurosteroid AP binds to all isoforms, but has enhanced sensitivity at the δ-subunit containing GABAA receptors. In the kindling studies, animals were tested for drug sensitivity 24 h after NSW. On the day of testing, animals were injected intraperitoneally with diazepam (0.1–1 mg/kg) or AP (1–10 mg/kg), 15 min before kindling stimulations. Control animals were injected similarly with vehicle (15% cyclodextrin). During each stimulation session, the behavioral seizure score and the AD duration were noted. Diazepam produced a dose-dependent suppression of behavioral seizure activity, and AD duration with significant effects at 0.1, 0.3, and 1 mg/kg in control (nonwithdrawal) animals, confirming diazepam protection against hippocampus kindling-induced seizures. In contrast, mice undergoing NSW had significantly decreased seizure protection by diazepam. At a dose of 1 mg/kg, diazepam produced an average of 95% and 30% decrease in seizure expression, in control and withdrawal groups, respectively. Taken together, these results are consistent with the notion that NSW causes relative insensitivity to diazepam caused by increased α4-containing GABAA receptor expression in the hippocampus. In addition, we investigated the efficacy of the prototype neurosteroid AP in control and NSW mice 24 h after NSW. Fully kindled control and neurosteroid-withdrawn mice were tested in the hippocampal kindling model with three doses of AP (1, 5, and 10 mg/kg s.c.). At these doses, AP exerted dose-dependent suppression of the behavioral seizures, and AD duration, in both control and NSW mice. Interestingly, NSW mice exhibited enhanced neurosteroid sensitivity, and greater AP-induced suppression of seizure severity, and AD duration, than in control mice. Moreover, plasma levels of AP achieved at various doses of AP treatment were similar between control and withdrawn groups,
especially without significant drug accumulation in withdrawn animals, indicating that AP sensitivity was not caused by pharmacokinetic factors (Reddy and Ramanathan, 2012). The synthetic neurosteroid ganaxolone (1, 3, and 10 mg/kg) also produced enhanced (60%) efficacy in fully kindled neurosteroid-withdrawn animals, confirming the enhanced sensitivity to neurosteroids in the NSW model of catamenial epilepsy (Reddy and Rogawski, 2001).
LIMITATIONS The validation of animal models of catamenial epilepsy requires certain criteria to be met for them to be representative of the human condition. They should have a close similarity in eliciting an epilepsy-like state, as well as a pathophysiology that mirrors the disease in humans. The NSW model partly meets these criteria and certainly offers some advantages over the use of conventional seizure models (Reddy, 2009). These models are based on similar physiological dynamics of ovarian P secretion during the menstrual cycle that cannot be simulated in the exogenous drug delivery models. Moreover, the 9-day elevation of P and AP levels in the rodent model closely matches the 10-day increase in AP levels during the luteal phase of the menstrual cycle. Although the exact etiology of catamenial epilepsy is not completely understood, the NSW model better simulates changes in the AP-to-estrogen ratio that is believed to be critical for perimenstrual catamenial epilepsy. However, the actual endocrine conditions that exist in the menstrual cycle are different from those observed in animal models of catamenial epilepsy. This is a major concern with most animal models developed in rodents. In rodents, the estrous cycle duration is 4–7 days, and the menstrual cycle in women lasts about 28 days.
INSIGHTS ON NEUROSTEROID WITHDRAWAL: WHAT THIS PARADIGM IS GOOD FOR Catamenial epilepsy affects a high proportion of women of reproductive age with drug refractory epilepsy. It is a multifaceted neuroendocrine condition (El-Khayat et al., 2008; Tuveri et al., 2008). Neurosteroids play a key role in the pathophysiology of catamenial epilepsy. Perimenstrual catamenial epilepsy is believed to be due to the withdrawal of neurosteroids as a result of the fall in P at the time of menstruation. There are currently no specific treatments for catamenial seizures. The NSW model of catamenial epilepsy has been used to investigate therapies for catamenial epilepsy. A key result is the observation that conventional antiepileptic drugs have reduced potency in protecting against catamenial seizures. This finding is consistent with women with catamenial epilepsy who do not respond to these drugs. The model therefore represents a
Perimenstrual Seizures and Neurosteroid Withdrawal Chapter | 62
form of the pharmacoresistance paradigm. The increased relative expression of benzodiazepine-insensitive GABAA receptors at the time of NSW likely accounts for the reduced activity of benzodiazepines. We unexpectedly found that neurosteroids, including their synthetic analogs, had enhanced activity in the perimenstrual catamenial epilepsy model (Reddy and Rogawski, 2000a,b, 2001). Based on our studies in catamenial epilepsy models, we suggested that “neurosteroid replacement” may be a useful approach to prevent catamenial seizure exacerbations (Reddy and Rogawski, 2009). A neurosteroid or a neurosteroid-like compound, such as ganaxolone, could be administered in a “pulse” prior to menstruation, and then withdrawn, or continuously administered throughout the month at low doses to avoid sedative side effects. Such low doses are expected to contribute little anticonvulsant activity during most of the menstrual cycle. Therefore, neurosteroids might provide an effective approach for catamenial epilepsy therapy. The molecular mechanisms underlying such enhanced neurosteroid sensitivity in catamenial epilepsy remain unclear. A δ-force hypothesis was coined to explain this phenomenon (Galanopoulou, 2015; Reddy, 2014). Recently, we found a novel plasticity of extrasynaptic δ-containing GABAA receptors in the dentate gyrus in a mouse perimenstrual model (Carver et al., 2014; Wu et al., 2013). In summary, the proposed mechanism poses that the perimenstrual decline in neurosteroids triggers the selective overexpression of α4δ subunits in the dentate gyrus granule cells, but this is not sufficient to suppress the increased seizure susceptibility of NSW mice in the absence of AP. Yet, this pathologic increase in α4δ subunits may act as a vehiclecatalyst (Trojan horse) for the exogenous AP to inhibit seizures. Indeed, this special sensitivity to AP that woman with perimenstrual epilepsy exhibit is in agreement with the findings from the NIH P trial, in which the responder group was women with the C1 type of catamenial epilepsy (Herzog et al., 2012). Interestingly, these women responders demonstrated significant posttreatment increase in AP levels (Herzog et al., 2014). This scenario would render perimenstrual epilepsy an ideal candidate to test the therapeutic effects of neurosteroids as a brief pulse treatment protocol during this perimenstrual period. Therefore, these findings may represent a molecular rationale for neurosteroid therapy of catamenial epilepsy.
ACKNOWLEDGMENT The author thanks Bryan Clossen and Victoria Golub for reading the manuscript.
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