Sex Differences in the Epilepsies and Associated Comorbidities: Implications for Use and Development of Pharmacotherapies

a. GABAergic chemoconvulsants

GABA antagonists are one of the most commonly families of chemoconvulsants in epilepsy research. Of this class of drugs, pentylenetetrazole (PTZ) remains a component of the National Institute of Neurological Disorders and Stroke–funded Epilepsy Therapy Screening Program (https://panache.ninds.nih.gov/CurrentModels.aspx). PTZ and the other GABA antagonists, in a dose-dependent manner, evoke absence-like generalized spike-and-wave discharges (SWDs), clonic seizures, and generalized tonic-clonic seizures. Surprisingly, although these drugs (PTZ, picrotoxin, bicuculline, etc.) share a common target, they differ in terms of sex differences (Table 1).

The threshold for picrotoxin-induced generalized tonic-clonic seizures is higher in male than female rats (Pericic et al., 1985; Schwartz-Giblin et al., 1989). Similarly, the latency to picrotoxin-induced akinetic seizures is also longer in males than females (Tan and Tan, 2001). This sex difference is also evident in cats, with female cats displaying a greater frequency of spinal motor neuron discharge than males following picrotoxin (Pericic et al., 1986). In mice, males showed increased sensitivity to picrotoxin compared with females (Pericic et al., 1986). In addition, mortality rates after picrotoxin are greater in male mice than female mice (Pericic et al., 1986) and are elevated in proestrous compared with estrous rats (Tan and Tan, 2001). Sex hormones influence picrotoxin responses, with estradiol treatment increasing sensitivity to pictrotoxin in male rats (Pericic et al., 1996). By contrast, testosterone increases susceptibility in females and decreases susceptibility in males (Tan and Tan, 2001). Consistent with the picrotoxin effects, female rats display greater sensitivity to allylglycine-induced convulsive seizures than do male rats (Thomas and Yang, 1991). Thus, in rats, females are more sensitive to picrotoxin than males, a pattern that is reversed in mice.

Interestingly, the threshold for PTZ-evoked clonic seizures is ∼10% lower in male rats compared with female rats (Kokka et al., 1992); this finding is somewhat counterintuitive when compared with the data on picrotoxin given that these drugs share a similar mechanism of action (i.e., both are GABA receptor channel blockers). In mice, PTZ responses are greater in females (Medina et al., 2001), and female mice display lower thresholds for response (Min et al., 2013), which is generally consistent with the effects of picrotoxin. Chronic administration of PTZ produces a progressive worsening of seizure severity (i.e., a kindling effect). Male and female rats acquire PTZ kindling at similar rates despite differing sensitivities to acute PTZ (Haeri et al., 2016).

As with PTZ, the threshold for myoclonus, running/bouncing clonus, and tonic hindlimb extension following intravenous bicuculline is lower in male rats compared with females (Finn and Gee, 1994; Pericic and Bujas, 1997). Similarly, males show lower thresholds than females to myoclonus, running/bouncing clonus, and tonic hindlimb extension induced by methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, a negative allosteric modulator of GABA_A receptors (Finn and Gee, 1994).

b. N-methyl-D-aspartate

Injection of the glutamate receptor agonist N-methyl-D-aspartate (NMDA) in rat pups primed by gestational betamethasone exposure is a common model of infantile spasms, and in this model, sex differences in seizures are not evident (Chachua et al., 2011), a pattern consistent with the clinical literature.

c. Electroshock

As with chemoconvulsant models, maximal electroshock remains a vital part of the screening pipeline for antiseizure medications. The endpoint of this model, tonic hindlimb extension, is particularly relevant for tonic and tonic-clonic seizures.

Female rats display lower maximal electroshock thresholds than do male rats (Woolley et al., 1961; Kokka et al., 1992). Consistent with this finding, a comprehensive analysis by sex in 10 strains found that females displayed, on average, a 10%–15% lower threshold for electroshock-evoked minimal, maximal, and psychomotor seizures (Frankel et al., 2001).

d. Inbred genetic models

Two of the most well established genetically epilepsy-prone rat (GEPR) models of epilepsy are the GEPR-3 and GEPR-9 strains. In response to acoustic stimulation, GEPRs display wild running, bouncing clonus, and, depending on the substrain, tonic extension. Neither the GEPR-3 nor the GEPR-9 substrains display sex differences in the ontogeny of audiogenic seizures (Reigel et al., 1989). However, in both the -3 and -9 strains, the penetrance of the audiogenic phenotype is greater in females than males (Kurtz et al., 2001). Moreover, female GEPR-9s display shorter latencies to seizures and more severe seizure scores than do age-matched GEPR-9 males (Mishra et al., 1988a,b). Inhibitors of adenosine metabolism cause heightened mortality in females compared with male GEPR-9s (Kommajosyula et al., 2016). Similarly, in the GEPR-3 strain, females display shorter latencies to wild running than do matched males (Mishra et al., 1989) and greater anticonvulsant responses to transient receptor potential cation channel subfamily V receptor 1 antagonists than males (Cho et al., 2018).

The Ihara’s Epilepsy Rat model displays a striking sex difference in penetrance; though essentially all male rats display tonic-clonic seizures, only ∼20% of females show similar responses (Amano et al., 1996). The Noda Epileptic Rat model also displays generalized tonic-clonic seizures with a high penetrance (>95%) in both sexes (Noda et al., 1998).

The El/Suz mouse (Suzuki and Nakamoto, 1982) is a selectively bred polygenic model of generalized epilepsy. These mice display handling-induced seizures and behavioral comorbidities (Bond et al., 2003; McFadyen-Leussis and Heinrichs, 2005) and are hyper-responsive to stress (Forcelli et al., 2007). Though the penetrance of the seizure phenotype does not differ as a function of stress in this strain (Leussis and Heinrichs, 2006), female El mice but not male El mice display increased cell density in the amygdala (Forcelli et al., 2007).

The DBA/1 inbred strain of mice has been used as a model of sudden unexpected death in epilepsy (SUDEP) (Faingold et al., 2010). In response to acoustic stimulation, these animals display tonic seizures, which end with hindlimb extension and respiratory arrest. Although the rate of SUDEP is higher in males than females in clinical populations (Hesdorffer et al., 2011b), the rate of SUDEP in DBA/1 mice is equivalent across sexes (Faingold and Randall, 2013).

e. Other genetic models

The explosion of genetic models of epilepsy over the last three decades makes a thorough characterization of effects difficult for several reasons. First, many models for the same “syndrome” exist, and in many cases, these models differ from one another. Second, differing genetic backgrounds of mice exert strong modulatory effects on seizure phenotype (Hawkins et al., 2016; Kang et al., 2018). Thus, only a few examples are provided below.

In the Brd2^+/− model of juvenile myoclonic epilepsy, males show reduced clonic seizure threshold, whereas females have reduced tonic-clonic threshold (Velíšek et al., 2011). Males and females also differ in open-field performance, with males displaying decreased anxiety-like behavior compared with wild-type littermates and females displaying increased anxiety-like behavior (Chachua et al., 2014). In models of Dravet syndrome (e.g., Scn1a^+/− mice), there have been reports of females showing greater mortality than males (Niibori et al., 2020) as well as reports of no sex differences (Kang et al., 2018). Therefore, more comprehensive screening for sex differences across genetic models is clearly needed.

a. Clinical studies

Psychiatric comorbidities are common in epilepsy (Josephson and Jetté, 2017) and estimated to occur in 25%–50% of patients (LaFrance et al., 2008), although higher incidence has been reported, with the highest prevalence observed in association with TLE (Bragatti et al., 2010) and drug-resistant epilepsy (depression = 55%; anxiety = 28.7%) (Kwon and Park, 2014; Jansen et al., 2019). The incidence of depression in epilepsy has been reported in 11%–80% of patients compared with 4.9%–17% in the general population (LaFrance et al., 2008). Similarly, the incidence of anxiety is higher in patients with epilepsy (15%–20%) (Brandt et al., 2010) compared with 5%–7% in the general population (LaFrance et al., 2008; Kanner, 2011; Kwon and Park, 2014; Brandt and Mula, 2016; Salpekar and Mula, 2018). It is important to note that the incidence of psychiatric comorbidities is even more prevalent in epilepsy as compared with other chronic illnesses such as cancer, diabetes, or asthma (Hermann et al., 1996). Furthermore, patients with psychiatric disorders, such as depression, are at a higher risk for developing epilepsy (Josephson and Jetté, 2017). Therefore, it is thought that psychiatric disorders and epilepsy may share a common underlying pathophysiological mechanism (Kanner, 2003).

Despite the high incidence of psychiatric comorbidities in epilepsy and the well established sex differences in anxiety and depression in the general population (Altemus et al., 2014), few studies have investigated sex differences in psychiatric comorbidities in epilepsy. The incidence of anxiety and depression is twice as common in females than in males in the general population (Altemus et al., 2014), and this is also true for patients with epilepsy. A retrospective study found that the incidence of depression was 15.5% in males and 26.8% in females with epilepsy (Chan et al., 2015). Similarly, anxiety disorders were found in 9.65% of males and 17.4% in females with epilepsy (Chan et al., 2015). These few studies suggest that there may be sex differences in psychiatric comorbidities in epilepsy. This represents a significant concern for epilepsy treatment given that psychiatric comorbidities are associated with worse epilepsy outcomes (Josephson and Jetté, 2017). Further studies are required to understand the extent of sex differences in psychiatric comorbidities in epilepsy and the impact on epilepsy outcomes.

b. Animal models

Similar to clinical studies, the majority of studies investigating sex differences in psychiatric disorders in experimental epilepsy models have focused solely on males, leaving us with limited information regarding sex differences in psychiatric comorbidities in preclinical epilepsy models. Existing studies suggest that there are sex differences in seizures in experimental animals as highlighted above [for review, see Scharfman and MacLusky (2006)]. In the few studies that have examined associated psychiatric comorbidities, increased anxiety- and depression-like behaviors have been observed in numerous experimental epilepsy models, including both genetic (Aguilar et al., 2018) and acquired epilepsy models (Mazarati et al., 2008; Becker et al., 2015; Hooper et al., 2018; Zeidler et al., 2018). However, none of these studies to date have demonstrated sex differences in anxiety- or depression-like behaviors in epilepsy models, although only a few have looked. In fact, Zeidler et al. (2018) state that no significant sex differences in depression- or anxiety-like behaviors were observed in chronically epileptic mice in the intrahippocampal KA model of epilepsy but provide the disclaimer that their experiments were not designed to test for sex differences. One study demonstrated subtle sex differences in behavior in a genetic epilepsy model, including differences in head dips off the open arms, an effect primarily driven by females (Aguilar et al., 2018). Some outcome measures also showed a main effect of sex but not a strain-by-sex interaction (Aguilar et al., 2018). Although overall these findings do not support sex differences in psychiatric comorbidities in epilepsy by using traditional behavioral tests and assessments of standardized outcome measures, there is an increased appreciation that males and females may exhibit different behaviors in these tests, and care should be taken in how to properly interpret these data (Shansky, 2018). Furthermore, the limited information on this subject is evident from this review, and additional studies are required for a better understanding of the mechanisms contributing to sex differences in comorbid psychiatric disorders in epilepsy.

a. Clinical studies

Both men and women with epilepsy are at increased risk for reproductive endocrine disorders compared with the general population (Herzog, 2008; Koppel and Harden, 2014). Up to 90% of men with epilepsy may present semen abnormalities, including low sperm count or abnormal sperm morphology or motility, and a population-based study quantified a 40% lower birth rate in men with epilepsy (Taneja et al., 1994; Artama et al., 2004; Herzog, 2008). It should be noted, however, that lower birth rates can be affected by many social factors (Mameniškienė et al., 2017) and are not necessarily a direct representation of fertility. Furthermore, changes in semen quality and sperm count are highly associated with the use of certain enzyme-inducing ASDs, such as valproic acid, carbamazepine, and oxcarbazepine (Roste et al., 2003; Isojarvi et al., 2004; Hamed et al., 2015; Ocek et al., 2018) and may thus be independent of epilepsy-induced changes. In women, common ailments include anovulatory menstrual cycles, oligomenorrhea (irregular cycle periodicity and infrequent menstruation), polycystic ovaries, and polycystic ovarian syndrome (PCOS) (Cummings et al., 1995; Bauer et al., 1998, 2000a,b; Morrell et al., 2002; Herzog et al., 2003a; Lofgren et al., 2007). Specifically, an estimated 10%–25% of women with TLE are diagnosed with PCOS compared with 4%–6% of the general population (Webber et al., 1986; Herzog and Schachter, 2001), and another 12% of women with TLE develop hypothalamic amenorrhea compared with 1.5% of the general population (Herzog et al., 1986b). Although much focus has been placed on these comorbidities in the context of TLE and other focal epilepsies, several studies have also documented these comorbidities in women with generalized epilepsy disorders (Morrell et al., 2002; Lofgren et al., 2007). In addition, although ASD treatment is highly associated with development of reproductive endocrine problems in both men and women (for examples, see Isojarvi et al., 1993, 2004; Murialdo et al., 1997; Harden, 2005a; Ocek et al., 2018), several lines of evidence indicate that seizure activity itself is a primary driver of these comorbid issues. In particular, multiple groups have documented a pattern in which women with TLE and seizures originating on the left side of the brain (i.e., left-sided TLE) show increased rates of PCOS, whereas women with TLE seizures originating in the right hemisphere (right-sided TLE) exhibit higher rates of hypothalamic amenorrhea (Herzog, 1993; Drislane et al., 1994; Herzog et al., 2003a,b; Kalinin and Zheleznova, 2007; Quigg et al., 2009). The findings indicate that distinct reproductive endocrine disorders may be related to the area of the brain primarily affected by seizure activity and suggest that specific neural pathways act as substrates to drive altered endocrine functions.

Investigations of the pathophysiology of reproductive endocrine comorbidities of epilepsy have naturally focused on the hypothalamic-pituitary-gonadal (HPG) axis, which links the brain to the control of fertility and reproduction in all mammalian species. Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus release the GnRH peptide to stimulate gonadotrope cells of the anterior pituitary to produce the gonadotropin hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). In both sexes, LH and FSH act on the gonads to induce gametogenesis and production of sex steroid hormones. LH and FSH can be readily detected in the peripheral bloodstream. Therefore, measurements of these hormones are commonly used as surrogate readouts of activity at the hypothalamic level of the HPG axis in humans, including in patients with epilepsy. An important feature of both LH and FSH is that these hormones are released in intermittent boluses, or pulses. Therefore, the pattern of pulsatility of these hormones as well as changes in mean basal levels are important indicators of disruptions to the hypothalamic-pituitary axis in people with epilepsy. In brief, several studies of both women and men with epilepsy have documented changes in the frequency of LH pulses (Meo et al., 1993; Drislane et al., 1994; Quigg et al., 2002; Herzog et al., 2003a), although others did not find a difference in LH pulsatility (Murialdo et al., 1995). Furthermore, impaired LH response to exogenous GnRH treatment is also commonly observed in both women and men with epilepsy (Herzog et al., 1982; Bilo et al., 1988; Murialdo et al., 1995), along with changes in mean LH or FSH levels (Bilo et al., 1988; Meo et al., 1993; Morrell et al., 2002; Herzog et al., 2003a). These changes in pituitary gonadotropin levels suggest that activity at the hypothalamic level, as well as further upstream in the brain, is altered such that pituitary response to GnRH and/or gonadotropin synthesis and release are commonly affected in both women and men with epilepsy, particularly TLE. These pathophysiological changes are challenging to assess in human patients but are beginning to be addressed in animal models.

As described in further detail below, the sex steroid hormones estradiol, progesterone, and testosterone exert strong effects on neural function with major implications for seizure activity. Therefore, disruptions to normal patterns of sex steroid synthesis and release can have profound impacts on seizure control. In this regard, it is notable that both women and men with epilepsy commonly show changes in circulating levels of these hormones. Although these changes often arise as side effects of treatment with ASDs, particularly valproic acid (Isojarvi et al., 1993, 2004; Harden, 2005a; Roste et al., 2005; Isojarvi, 2008; Ocek et al., 2018), epilepsy and seizure activity have also been independently associated with changes in hormone levels, particularly regarding testosterone. In men, for example, decreased testosterone has been documented in men with TLE, with recovery to normal levels often seen after temporal lobe resection and improved seizure control (Bauer et al., 2000c, 2004). It should be noted, however, that not all studies have found changes in testosterone in male patients (Mikkonen et al., 2004). Furthermore, testosterone may be lower in men with TLE compared with men with seizure foci outside of the temporal lobe (Bauer et al., 2004), suggesting specific etiologies related to downstream hormone changes dependent on the neural circuits primarily affected by the seizure activity. Conversely, elevated testosterone is often observed in women with TLE (Herzog et al., 2003a) or idiopathic generalized epilepsy (Lofgren et al., 2007). Particularly interesting findings were that women with left-sided TLE showed higher testosterone compared with women with right-sided TLE (Herzog et al., 2003a) and that women with right-sided TLE showed significantly decreased estradiol levels compared with women with left-sided TLE (Herzog et al., 2003b). These results further suggest that downstream hormone changes can reflect impairments and disruption to specific neural circuits.

Another pituitary hormone critical to proper regulation of HPG axis function is prolactin (PRL), which is produced under the control of dopaminergic synaptic inputs projecting from the hypothalamic arcuate nucleus into the posterior pituitary. This direct synaptic connection is thus anatomically poised to provide an uninterrupted link between seizure activity in the brain and altered pituitary PRL release. Because elevated PRL levels suppress the GnRH-LH axis, hyperprolactinemia can be another mechanism of impaired reproductive endocrine function in patients with epilepsy. With respect to changes in PRL in women with epilepsy, some studies have documented increased levels (Bilo et al., 1988), whereas others have not found baseline differences (Dana-Haeri et al., 1984; Herzog et al., 2003a). Acute postictal increases in prolactin have also been reported in women with epilepsy (Collins et al., 1983; Dana-Haeri et al., 1983; Pritchard et al., 1985). Approximately 10% of men with TLE may show signs of hyperprolactinemia (Herzog et al., 1986a), although other studies have documented acute increases in PRL immediately after seizure activity without a change in mean levels (Abbott et al., 1980; Collins et al., 1983; Dana-Haeri et al., 1983; Pritchard et al., 1985; Quigg et al., 2002). Still other studies documented similar postictal increases in PRL, but the sex of the subjects was not specified (Trimble, 1978; Sperling et al., 1986). Therefore, seizure-associated changes in PRL levels may be seen in both men and women with epilepsy, particularly TLE. Indeed, acute measurement of serum PRL postictally has been suggested as a possible diagnostic tool to distinguish epileptic seizures from psychogenic nonepileptic seizures (Chen et al., 2005a).

In addition to impairments of reproductive endocrine systems, many men and women with epilepsy also complain of sexual dysfunction. Although some studies have failed to find higher rates of such complaints in people with epilepsy (Jensen et al., 1990), others have documented a variety of symptoms [for comprehensive reviews, see Harden (2005b), Luef and Madersbacher (2015)]. For example, several studies have documented hyposexuality, decreased sexual drive, and lower arousability in substantial proportions of men and women with epilepsy (Demerdash et al., 1991; Christianson et al., 1995; Murialdo et al., 1995; Morrell and Guldner, 1996; Daniele et al., 1997; Duncan et al., 1997; Silveira et al., 2001; Baird et al., 2003; Kuba et al., 2006; Henning et al., 2019). Furthermore, deficits in arousability, genital blood flow, and erectile function appear to reflect physiologic impairments (Morrell et al., 1994; Guldner and Morrell, 1996; Morrell and Guldner, 1996). Disproportionate rates of sexual dysfunction observed in patients with right-sided TLE compared with left-sided TLE also suggest primary neural components drive these comorbidities (Daniele et al., 1997; Herzog et al., 2003b) in addition to more frequent observations of temporal lobe seizure foci in women with epilepsy that exhibit sexual dysfunction compared with patients that do not exhibit the comorbidity (Demerdash et al., 1991). It should be noted, however, that sexual dysfunction can also be associated with ASD treatment (Herzog et al., 2005; Luef and Madersbacher, 2015), although some studies have not observed such an effect (Henning et al., 2019). In summary, various presentations of sexual dysfunction are commonly observed in both men and women with epilepsy.

b. Animal models

The investigation of reproductive endocrine comorbidities of epilepsy is one of the few areas of preclinical research in which the studies investigating female animals far outnumber those examining males. This is primarily due to the commonalities between the estrous cycle of female rodents and menstrual cycle in humans [Fig. 1; see also Fig. 2 in Scharfman and MacLusky (2014b)] such that disruptions to the estrous cycle are useful experimental models with which to investigate the pathophysiological mechanisms of reproductive endocrine disorders in epilepsy. In this regard, female rodents tested in various models of epilepsy have also shown high propensity for developing disrupted estrous cycles and other indicators of reproductive endocrine dysfunction. For example, female Wistar rats tested in the intrahippocampal KA model of TLE were found to spend decreased time in the stages of proestrus and estrus and increased time in metestrus (Amado et al., 1987). In recent studies examining female mice in a similar intrahippocampal KA model, prolongation of estrous cycle period, typically characterized by more time spent in diestrus, was observed in the majority of mice (Li et al., 2016, 2018). Furthermore, these differences did not appear for at least 6 weeks after the KA injection, indicating that it was the chronic epileptic condition, and not the acute effects of KA excitotoxicity, that drove the cycle disruption (Li et al., 2016). In studies employing the pilocarpine post-SE model of TLE, Wistar rats showed more time spent in diestrus from 2 to 6 weeks after injection (Amado and Cavalheiro, 1998), and Sprague-Dawley rats showed a variable response, with one-third showing immediate cessation of cycling after pilocarpine-induced SE, one-third developing irregular cycles over time, and the remaining animals maintaining regular cyclicity (Scharfman et al., 2008). Disrupted estrous cycles have also been described in pilocarpine-treated mice (Fawley et al., 2012). Lastly, amygdala kindling models have also been linked with estrous cycle disruption, producing increased time in estrus and/or decreased time in diestrus in Sprague-Dawley and Wistar rats in some studies (Edwards et al., 1999d; Pan et al., 2013) and cycle period lengthening without induction of persistent estrus in others (Hum et al., 2009). In summary, these studies indicate that disruptions to the estrous cycle are commonly observed in rodent models of epilepsy.

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Fig. 1.

Changes in seizure susceptibility associated with fluctuations in ratios of estradiol (E, pg/ml) and progesterone (P, ng/ml) levels in the human menstrual and rodent estrous cycles. (A) E:P (solid line) and P:E (dashed line) ratios in the human menstrual cycle and associated catamenial seizure clustering. Hormone data are adapted from Thorneycroft et al. (1971). See section III. Catamenial Epilepsy: Fluctuations in Seizure Occurrence across the Menstrual Cycle for discussion of catamenial seizure clustering. (B) E:P and P:E ratios in the rat estrous cycle. Hormone data are adapted from Smith et al. (1975). (C) E:P and P:E ratios in the mouse estrous cycle. Hormone data are adapted from Walmer et al. (1992). See section IV.B.1. Estrous Cycle–Associated Changes in Seizure Susceptibility for discussion of changes in seizure susceptibility associated with the estrous cycle in rats and mice.

Changes in pituitary gonadotropin (LH and FSH) release or content, as observed in human patients, have also been documented in several rodent models of chronic epilepsy and acute seizure induction in both males and females (Bhanot and Wilkinson, 1982; Fujii et al., 1984; Amado and Cavalheiro, 1998), although changes in LH pulsatility patterns have not yet been tested in either sex. These changes appear to be at least partially due to paroxysmal activity in limbic structures, as several studies have shown changes in LH and/or FSH release following electrical stimulation of hippocampus and/or amygdala in both male and female animals (Velasco and Taleisnik, 1969; Gallo et al., 1971; Kawakami et al., 1973a,b).

The major advantage of animal models is increased direct access to the hypothalamic components of the HPG axis, particularly the GnRH neurons. In this regard, earlier studies limited to immunocytochemical staining for the GnRH peptide yielded conflicting results, with decreased GnRH immunoreactivity found in female rats after intra-amygdalar injection of KA or systemic pilocarpine induction of SE (Amado et al., 1993; Friedman et al., 2002) but no difference in GnRH staining in pilocarpine-injected female mice (Fawley et al., 2012). A recent study, however, provided the first direct documentation of functional changes in GnRH neuron activity and excitability, showing that in female mice, the rate and pattern of GnRH neuron firing was altered in the intrahippocampal KA model of TLE and that the direction of change in KA-injected mice compared with controls shifted from diestrus to estrus. Specifically, GnRH neurons from KA-injected mice showed higher firing on diestrus and suppressed firing on estrus, and these differences were most profound in the mice that concomitantly showed prolonged cycle length. Moreover, male mice tested in the same model showed a more modest effect, with only a subset of GnRH neurons (those with cell bodies in the medial septum) displaying elevated firing activity (Li et al., 2018) (Fig. 2).

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Fig. 2.

Sex differences in impacts of epilepsy on GnRH neuron firing activity. Mean ± S.E.M. for mean firing rate of GnRH neurons from saline-injected controls (white bars) and KA-injected female mice recorded on diestrus and on estrus (A) and male mice (B). KA-injected females are divided into KA-long (red bars) and KA-regular (blue bars) groups based on estrous cycle length (long = ≥7 days, regular = 4–6 days). Cells are classified based on anatomic location of somata in medial septum (MS), preoptic area (POA), and anterior hypothalamic area (AHA). Adapted from Li et al. (2018).

As in human patients with epilepsy, several animal studies have documented changes in circulating levels of the sex steroids in preclinical models of TLE, with variations dependent on the species and specific epilepsy induction model (Table 2). For example, amygdala-kindled female Wistar rats that became acyclic developed higher baseline estradiol levels along with higher estradiol and testosterone levels following progesterone treatment (Edwards et al., 1999d). Decreased progesterone levels were also documented in female rats tested in the intrahippocampal KA model of TLE (Amado et al., 1987). Sprague-Dawley rats treated in the pilocarpine post-SE model of TLE, however, did not show differences in estradiol or progesterone when measured on diestrus but did show elevated testosterone (Scharfman et al., 2008), whereas Wistar rats tested in the pilocarpine model displayed increased estradiol and decreased progesterone (Amado and Cavalheiro, 1998). By contrast, in a recent study using the intrahippocampal KA model of TLE in female mice, the pattern of change in estradiol or progesterone levels as measured 2 months after KA injection changed with estrous cycle stage and depended on whether the mice developed prolonged, disrupted estrous cycles. Specifically, progesterone levels were suppressed in KA-injected mice that developed long cycles but not in KA-injected mice that retained normal cycle periodicity, and this pattern was observed on both diestrus and estrus. Regarding estradiol, however, no differences were observed between groups in levels of this hormone on diestrus, but KA-injected mice, independent of estrous cycle period, displayed elevated levels compared with controls (Li et al., 2018). With respect to male animals, in one study, amygdala-kindled rats appeared to show increased testosterone, but acute maximal electroshock-induced seizures transiently decreased testosterone levels (Edwards et al., 1999a). These findings suggested that focal and generalized seizures may have different downstream impacts on testosterone production and/or metabolism. Male mice treated in the intrahippocampal KA model of TLE, however, did not show a change in testosterone levels when measured 2 months after KA injection (Li et al., 2018). So far, PRL has received less attention in studies of animal models of epilepsy, with conflicting reports of higher PRL levels described in amygdala-kindled female rats (Edwards et al., 1999d), decreased levels observed in male rats following acute electrical stimulation in the amygdala (Kawakami et al., 1973a), and no change observed in pilocarpine-treated female rats (Scharfman et al., 2008). In summary, although the specific directions and degrees of change in sex steroid levels may display species differences and vary across epilepsy models, these effects may have important implications for understanding and interpreting features of epileptiform activity that are both distinct between and common to male and female animals.

To date, sexual dysfunction in animal models of epilepsy has received less investigatory emphasis than reproductive endocrine disorders. In one study, male cats displayed reduced sexual behavior for at least 6 months following intra-amygdalar injection of aluminum hydroxide to induce focal epilepsy (Feeney et al., 1998). In studies using the pilocarpine post-SE model of TLE in Wistar rats, males showed longer latency to initiate sexual activity and reduced numbers of mounts and intromissions, and females displayed reduced sexual interest in a male rat (proceptivity) and suppressed receptivity (readiness to allow copulation as exhibited by the lordosis response) following estradiol and progesterone priming treatment sufficient to induce estrus in control rats (Andersen et al., 2012; Alvarenga et al., 2013). Therefore, in line with the common observation of hyposexuality in people with epilepsy, animal models also appear to display at least some degree of sexual dysfunction in both sexes. Given the increased feasibility of measuring sex behavior in rodent models of epilepsy in the absence of confounding antiepileptic drug treatment, further investigation in this area is likely to yield insights into physiologic links between epileptiform and seizure activity and altered sexual function.

a. Clinical studies

A large body of work has accumulated evidence for functional sex differences in human cognition and various forms of learning and memory, at least when assessed on average, although the specific features and underlying mechanisms are a matter of ongoing debate (Andreano and Cahill, 2009; Hamson et al., 2016; Asperholm et al., 2019). As many forms of epilepsy reflect seizure activity and, in some cases, pathologic damage to brain regions that participate in these cognitive functions, it is not surprising that cognitive and learning deficits are major comorbidities of epilepsy. Comprehensive discussion of the links between epilepsy and cognition across the lifespan, and the various forms of memory and neurocognitive function that can be affected, are beyond the scope here and are available in many recent reviews (Bell et al., 2011; Lin et al., 2012; Helmstaedter and Witt, 2017; Semple et al., 2019). It should be noted as well that there is growing appreciation for a comorbid relationship between epilepsy and Alzheimer disease, as discussed in other recent reviews (Chin and Scharfman, 2013; Sen et al., 2018). Assessing sex differences in the relationship between epilepsy and Alzheimer disease, however, is somewhat complicated by differential incidence and progression of Alzheimer disease and other forms of aging-related dementia in women and men (Hebert et al., 2013; Pike, 2017; Ferretti et al., 2018). The documented increase in risk for Alzheimer disease in postmenopausal women compared with age-matched men (Ferretti et al., 2018), however, underscores the need to identify causal mechanisms that produce sex-specific outcomes as well as features common to both sexes. Such insights will require studies aimed at understanding the mechanisms of sex differences in epilepsy and Alzheimer disease separately as well as the comorbid interactions between the two neurologic disorders in the context of both aging-dependent and -independent changes. In this section, we will focus on those studies that suggest sex differences in the presentation of cognitive comorbidities in children and adults, without the confounds of dementia-related disorders that could lead to cognitive impairment independently of the effects of epilepsy and seizures. Furthermore, we will focus particularly on those studies that specifically assessed and described whether a sex difference was observed.

Children with epilepsy are at particular risk for cognitive impairments and learning deficits, and sex can play a role in the presentation and features of these comorbidities. For example, in a study of children and adolescents with intractable epilepsy examining impairments in episodic verbal and visual memory, girls exhibited better delayed recall of stories and learning of a word list, but no sex difference was observed in performance on delayed recall of words or in visual tasks (Smith et al., 2009). Both boys and girls with epilepsy exhibit higher rates of autism, intellectual disability, and attention-deficit/hyperactivity disorder compared with children in the general population (Socanski et al., 2013; Aaberg et al., 2016; Williams et al., 2016). Although some studies document higher numbers of boys with epilepsy affected than girls (Aaberg et al., 2016), these findings may reflect an underlying male bias of these neurodevelopmental disorders in the general population, independent of specific effects of epilepsy and seizure activity (Rucklidge, 2010; Baron-Cohen et al., 2011; May et al., 2019). Other studies, however, have found similar rates of attention-deficit/hyperactivity disorder in both boys and girls with epilepsy [for review, see Williams et al. (2016)]. In addition, an analysis of data on children with epilepsy from the National Longitudinal Survey of Children and Youth in Canada found a slight preponderance of learning disabilities in girls (Prasad et al., 2014). The outcomes of epilepsy in children are also of interest given the increased potential for plasticity and functional compensation early in development. In this regard, it is interesting that patients who exhibit left-sided temporal lobe seizures by 1 year of age, when examined in early adulthood, can display sex-specific outcomes in cognitive impairment, and this effect is influenced by the lateralization of language function. Specifically, males were found to show generalized cognitive impairment of both verbal and nonverbal functions, independent of whether the left hemisphere remained dominant for speech. This effect contrasted with the outcomes in female patients examined, in which only some verbal functions were impaired, but nonverbal functions were largely unaffected if the left hemisphere remained dominant for speech (Strauss et al., 1992). Altogether, these and other studies suggest that neurobehavioral outcomes are prominent comorbidities in children with epilepsy, that these comorbidities can exhibit sex-specific features or prevalence, and that the long-lasting outcomes later in life can continue to exhibit sex differences in presentation and/or underlying mechanism.

Neuropsychological testing of adults with epilepsy has yielded interesting insights into sex differences in various forms of learning and memory, both in relation to epilepsy and to human cognition in general. To a large extent, and perhaps as to be expected, most of the focus in this domain has been in studying patients with TLE. In general, most studies indicate that women with epilepsy typically display better verbal memory than men with epilepsy (Berenbaum et al., 1997; Helmstaedter et al., 1999, 2004; Berger et al., 2017, 2018), although it should be noted that not all studies show a sex difference (Davies et al., 1998). After surgical resection of epileptogenic tissue of the anterior temporal lobe, women often continue to display superior performance (Trenerry et al., 1995; Berenbaum et al., 1997; Davies et al., 1998; Bengtson et al., 2000; Bjornaes et al., 2005; Berger et al., 2017). Overall, therefore, it appears that women in general display better performance on verbal learning and memory tests, and this sex difference confers a degree of resilience to decline in these measures in the face of epilepsy. Similarly, women without epilepsy, women with TLE, and women with generalized epilepsy all show better delayed face recognition memory when compared with men in these respective groups (Bengner et al., 2006). With regard to spatial memory as measured by hippocampal activation during an object location memory task in a virtual environment, it appears that in healthy controls, women display a left-lateralized activation pattern, whereas men display a right-lateralized pattern (Frings et al., 2006). In patients with TLE, by contrast, the lateralization pattern in this same task appears determined by the side of the seizure focus, not sex (Frings et al., 2008). Taken together, these studies suggest that various forms of memory and/or the organization of underlying neural substrates for these functions are changed in epilepsy, with impacts and features reflecting the sex of the patient.

b. Animal models

As would be expected from the observations of prominent cognitive comorbidities in patients with epilepsy, similar changes have also been well documented in various animal models of epilepsy. For a recent comprehensive review of behavioral comorbidities in preclinical models of epilepsy, see Holmes (2015). As is the case regarding other aspects of preclinical studies of epilepsy and seizure activity, however, few studies have specifically investigated the potential impact of sex as a biologic variable on these measures. Two recent studies of different genetic mouse models, however, have yielded some interesting sex-specific differences. First, the EL mouse, which displays autism-like behavioral features and comorbid epilepsy, appears to respond to ketogenic diet treatment in a sex-specific manner, with females showing greater improvement in sociability and stronger reduction in stereotyped repetitive self-grooming behavior (Ruskin et al., 2017). In addition, a recent study investigating heterozygous potassium voltage-gated channel subfamily Q member 2 knockout mice, which do not show spontaneous seizures but do show a reduced seizure threshold, observed male-specific increases in compulsive marble-burying and social dominance (Kim et al., 2020). By contrast, a recent study investigating both males and females in the GEPR-3 rat model documented that these rats display impaired novel object recognition, and a greater proportion of animals failed to explore the objects compared with Sprague-Dawley control counterparts, but it did not observe a sex difference in this effect (Aguilar et al., 2018).

With respect to pharmacological models of seizure induction and epilepsy, one study examining the behavioral consequences of KA-induced SE in newborn (4–6 days of age) rat pups documented that males showed stronger deficits in spatial learning as tested in a Barnes maze at 16–19 days of age than did the corresponding female pups (Akman et al., 2015). Conversely, however, in another study in which newborn rat pups were exposed to subconvulsive doses of domoic acid [which can lead to behavioral abnormalities suggestive of focal seizures as well as a lowering of seizure threshold (Gill et al., 2010)], the female, but not male, rats showed impaired spatial learning in a Morris water maze (Doucette et al., 2007). A series of studies all testing C57BL/6J mice have also documented varying effects. For example, one study of adult mice tested 2 months after pilocarpine-induced SE showed male-specific impairments in object exploration and reduced immobility in a forced swim test but no sex differences on other parameters, including open-field exploration and object recognition (Oliveira et al., 2015). Another recent study examining the short-term effects (within 5 days) of systemic KA induction of SE documented that male mice showed worse performance than females on novel object recognition, but it should be noted that the males also showed increased seizure susceptibility in response to the KA injection (Li and Liu, 2019). Therefore, the sex differences in behavioral consequences may simply reflect sex differences in the initial seizure severity. Conversely, direct unilateral injection of KA into the hippocampus can produce deficits in spatial memory in mice in a manner that does not appear to differ between males and females (Zeidler et al., 2018).

Although most investigations have focused on the neurobehavioral consequences of epilepsy and seizure activity, one recent study provides an intriguing potential sex-specific link between underlying performance on the Morris water maze in control conditions and subsequent seizure susceptibility. When Wistar rats were tested first in the Morris water maze and then challenged with PTZ seizure induction, the female rats displayed a correlation between maze performance and later seizure susceptibility that was absent in the males, with better maze performance correlating with reduced subsequent seizure susceptibility (Haeri et al., 2016). Altogether, only a few studies of animal models of epilepsy document sex differences in cognitive comorbidities, and others document the lack of a sex difference in a given behavioral parameter tested. However, there have not been sufficient studies performed to suggest that sex differences in cognitive comorbidities in animal models are largely absent. Systematic inclusion of both male and female animals, and formal statistical testing for the presence of any sex differences, would greatly help to improve our understanding of which cognitive comorbidities, if any, show a sex-specific predominance.

a. Clinical studies

In addition to the prominent psychiatric, reproductive, and cognitive comorbidities described in the previous sections, people with epilepsy are also at increased risk for a variety of other somatic conditions. Among these, urinary tract infection, hypothyroidism, and migraine are in the top 10 conditions for women, whereas cancer, coronary artery disease, and gastroesophageal reflux disease are in the top 10 for men (Wilner et al., 2014). Of these, the best studied is migraine, which also exhibits a higher prevalence in women in the general population worldwide (Stewart et al., 1992, 2008; Sakai and Igarashi, 1997; Vetvik and MacGregor, 2017).

The relationship between migraine and epilepsy is of particular interest for several reasons. First, both are episodic disorders (Haut et al., 2006; Rogawski, 2012); second, migraine is the most common type of headache observed in patients with epilepsy (Mainieri et al., 2015); third, the presence of comorbid migraines can negatively impact the prognosis of becoming seizure-free (Velioğlu et al., 2005); and fourth, many ASDs are effective as prophylactic treatments for migraines, producing a reduction in the number of episodes (Sprenger et al., 2018). Of note, migraine is far more commonly observed in patients with epilepsy than epilepsy is observed in migraine sufferers, simply because migraine on its own is far more prevalent (Bigal et al., 2003). Several studies have documented higher rates of migraine in people with epilepsy (for review, see Bigal et al., 2003; Haut et al., 2006; Rogawski, 2012), although there are exceptions (Brodtkorb et al., 2008; Tonini et al., 2012). Intriguingly, however, the sex difference in migraine prevalence observed in the general population appears to be largely equalized in the face of epilepsy. Whether focusing on acute postictal headache or peri-ictal headache, or migraine in general, relatively similar rates have been observed in both men and women with epilepsy (Forderreuther et al., 2002; Karaali-Savrun et al., 2002; Ito et al., 2004; Syvertsen et al., 2007; Mameniskiene et al., 2016), and the sex of the patient does not appear to influence the type of headache (e.g., migraine, tension, or unclassified) associated with seizure activity (Leniger et al., 2001). This does not exclude the possibility, however, that the underlying mechanisms and triggers of migraine and other headaches in epilepsy may be different between men and women.

b. Animal models

There is growing investigation of the underlying neurobiological mechanisms of migraine in animal models (for review, see Bolay et al., 2011; Ferrari et al., 2015; Pavlovic et al., 2017). Most of the focus in animal models has been on measures of cortical spreading depression (CSD), which is postulated to underlie the migraine aura (Charles and Baca, 2013). In this regard, it is intriguing that CSD susceptibility in rodent models also exhibits certain sex differences. For example, the threshold for triggering CSD in wild-type C57BL/6 mice appears to be lower in females, in both the potassium chloride and tetanic stimulation models of CSD induction (Brennan et al., 2007). Furthermore, mouse models of familial hemiplegic migraine type 1, which harbor mutations in the Cacna1a gene encoding the α1A subunit of Cav2.1 channels, exhibit sex differences, with females exhibiting increased susceptibility to CSD and neurologic motor deficits than males and increased propagation of CSD into subcortical structures (Eikermann-Haerter et al., 2009, 2011).

In terms of potential links between migraine and epilepsy, only a handful of studies have addressed this issue in animal models. Nevertheless, a study of Wistar audiogenic rats indicated intriguing sex differences that reversed with exposure of the rats to audiogenic kindling stimulation. Specifically, when tested prior to audiogenic kindling, the female and male rats showed higher and lower CSD conduction velocity, respectively, compared with controls of the same sex. After audiogenic kindling, however, the female rats showed lower CSD propagation than controls, whereas males showed higher CSD propagation (Guedes et al., 2009). Conversely, mice that harbor heterozygous or homozygous knockout mutations of proline-rich transmembrane protein 2, which underlie a group of disorders including epilepsy and migraine, do not appear to exhibit sex differences in PTZ or audiogenic seizure induction or in different motor or cognitive behaviors (Michetti et al., 2017). Lastly, a pair of recent studies examining a mouse model of familial hemiplegic migraine type 2, a subtype of severe migraine with aura and comorbid epilepsy, have documented sex differences in some parameters but not others. These mice, which carry a mutation in the astrocyte-specific α2-isoform of the Na⁺/K⁺ ATPase, may exhibit sex differences in certain behavioral comorbidities, including open-field exploration, and elevated cortical and hippocampal glutamate levels in adult females (Bottger et al., 2016). In tests of CSD susceptibility, a sex difference was not observed in young mice, but susceptibility was lowered specifically in aged, reproductively senescent females; however, these mice do not appear to show a sex difference in susceptibility to epileptiform activity (Kros et al., 2018). Altogether, the few studies that have accounted for seizures or epilepsy have primarily focused on models of rare, more severe subtypes of migraine. Therefore, it is unclear how the findings may relate more broadly to heterogeneous epilepsies and to the milder forms of migraine observed more generally in people with epilepsy.

a. Estradiol

Proconvulsant effects of estradiol have been observed across diverse animal models of seizure susceptibility (Table 3). Estradiol facilitates chemoconvulsant PTZ kindling in ovariectomized female rats (Hom and Buterbaugh, 1986). Estradiol decreases the latency to KA-evoked status in ovariectomized rats (Woolley, 2000) but interestingly decreases post-status cell loss (Reibel et al., 2000). Estradiol also reduces electroshock seizure threshold (Woolley and Timiras, 1962c) and accelerates both hippocampal and amygdala kindling (Edwards et al., 1999c). Similarly, in ovariectomized females, estradiol facilitates kindling from the anterior neocortex (Buterbaugh, 1989), dorsal hippocampus (Buterbaugh and Hudson, 1991), and amygdala (Hom and Buterbaugh, 1986; Buterbaugh, 1987). Estradiol also facilitates amygdala kindling in male rats (Saberi and Pourgholami, 2003). It should be noted, however, that some anticonvulsant effects of estradiol have also been observed. For example, in ovariectomized rats, the latency to NMDA-evoked seizures can be increased by estradiol (Vathy et al., 1998). In addition, ovariectomy results in an increased sensitivity to NMDA marked by longer total seizure duration and greater numbers of seizures than control animals; this effect is normalized by estradiol (Kalkbrenner and Standley, 2003).

b. Progesterone

Progesterone has primarily anticonvulsant and antiepileptic properties in animals and humans, although in a randomized placebo-controlled trial, progesterone was primarily effective in treating women with a perimenstrual catamenial pattern only (Jacono and Robertson, 1987; Herzog, 1999; Reddy, 2009). Progesterone has long been known to have antiseizure activity in a variety of animal models of epilepsy (Table 3). In recent years, numerous studies have confirmed the powerful anticonvulsant activity of progesterone in diverse animal seizure models (Reddy et al., 2004, 2010). For example, progesterone suppresses PTZ-evoked seizures in female mice (Frye et al., 2002) and protects against NMDA-evoked seizures in male mice (Członkowska et al., 2000). Consequently, seizure susceptibility is typically low during physiologic conditions associated with high progesterone. In women with epilepsy, natural cyclic variations in progesterone during the menstrual cycle could influence catamenial seizure susceptibility, as detailed above. It should be noted, though, that progesterone receptor (PR) agonists may increase the number of spontaneous seizures in female chronically epileptic rats (Shiono et al., 2019).

Progesterone acts on the brain through two different mechanisms. The first pathway involves binding to PRs and exerting effects through both genomic and nongenomic mechanisms. The second pathway involves modulating GABA_A receptors via synthesis of neurosteroids (Fig. 3). Neurosteroids rapidly alter neuronal excitability through direct interaction with GABA_A receptors (Macdonald and Olsen, 1994; Belelli et al., 2002). In the process of neurosteroidogenesis, progesterone is converted to allopregnanolone by sequential A-ring reductions. Another neurosteroid, tetrahydrodeoxycorticosterone (THDOC), is produced by reduction of deoxycorticosterone. It was demonstrated that PRs require longer periods of time to exhibit their effects, whereas neurosteroid synthesis via progesterone conversions occur rapidly, suggesting that these conversions could potentially be more relevant to developing pharmaceutical treatments. This hypothesis was supported by comparing wild-type and PR knockout mice to show that progesterone can still produce anticonvulsant effects in mice that lack PRs. Blockade of progesterone conversion to neurosteroids by finasteride treatment prevented the anticonvulsant effects, indicating that these outcomes were mediated by allopregnanolone (Reddy et al., 2004).

Because endocrine fluctuations in plasma levels of progesterone and other steroids can mediate neurosteroid availability, there are apparent differences between sexes concerning concentrations of neurosteroids in the brain. Though neurosteroids are capable of shaping inhibition and producing behavioral effects in both males and females, the regulation of neurosteroid activity may be sex-specific (Gulinello and Smith, 2003). Differences in maximal GABA_A receptor potentiation are observed between male and female rats for THDOC but not for allopregnanolone or androgenic neurosteroids (Wilson and Biscardi, 1997). Sex differences in the expression of 3α-hydroxysteroid dehydrogenase are evident during puberty but subside in the brain as it matures into adulthood; sex-specific gonadal and adrenal endocrine activity have a significant effect on the ability of allopregnanolone to modify anxiolytic (i.e., anxiety-reducing) actions based on variations in biosynthesis of steroid hormones (Mitev et al., 2003). Sex differences are evident in the anticonvulsant activity of neurosteroids; however, the potential mechanisms remain unclear. It is likely that differences in post-synaptic or extrasynaptic GABA_A receptor expression and function may underlie the sex differences in seizure sensitivity and the anticonvulsant activity of neurosteroids (Reddy et al., 2019) (Fig. 4). In this regard, progesterone has also anecdotally been reported to increase typical absence seizures in humans (Grunewald et al., 1992). Moreover, allopregnanolone, which is synthesized from progesterone, significantly increases SWDs in WAG/Rij rats (Budziszewska et al., 1999), consistent with postulated roles for neurosteroid-sensitive extrasynaptic GABA_A receptors in the thalamus in driving absence seizure activity (Banerjee and Snead, 1998; Cope et al., 2009; Errington et al., 2011).

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Fig. 4.

Sex differences in allopregnanolone potentiation of tonic GABAAR-mediated currents in hippocampal dentate gyrus granule cells (DGGCs) in wild-type (WT) and δ GABAAR subunit knockout mice (δKO). (A) Representative tonic current recordings from DGGCs in slices from WT male and female mice. Allopregnanolone (AP, 0.1–1.0 µM) was applied to the bath in addition to 1 μM GABA to measure allosteric enhancement of tonic current. (B) Summarized data of AP concentration response from male and female DGGCs in WT mice. (C) Representative tonic current recordings in the presence of AP or/and GABA from DGGCs in slices from δKO mice. (D) Summarized data of AP concentration response from male and female DGGCs in δKO mice. Tonic current was normalized to cell capacitance (pA/pF) as a measure of current density. The GABA-A receptor tonic current was expressed as the outward shift in holding current after the application of gabazine (50 mM). *P < 0.05 vs. males; #P < 0.05 vs. 1 µM GABA baseline current in the same sex (n = 6−8 cells per group). Adapted from Reddy et al. (2019).

c. Testosterone

Testosterone is synthesized by both the testes and the ovaries and, to a much lesser degree, the adrenal gland, and it can also be synthesized de novo in the brain from cholesterol. Testosterone is converted in the brain to estradiol by aromatase or to the nonaromatizable androgen dihydrotestosterone (DHT) by 5α-reductase (Meinhardt and Mullis, 2002; Swerdloff et al., 2017). The aromatase and 5α-reductase enzymes are expressed by both neurons and glia, although there are regional differences in which cell types are the dominant sources of each enzyme (MacLusky et al., 1987; Martini et al., 1993; Zwain et al., 1997; Melcangi et al., 1998; Zwain and Yen, 1999; Hojo et al., 2004). Castration of male rats as well as testosterone replacement produces both pro- and anticonvulsant effects in the model depending on timing and duration of treatment (Woolley and Timiras, 1962b) (Table 3). On balance, it appears that the actions of estradiol on neuronal excitability and seizure susceptibility are opposite those of DHT, with estradiol and DHT increasing and decreasing these parameters, respectively. Accordingly, aromatase inhibition has demonstrated some efficacy in improving seizure control in men with epilepsy (Harden and MacLusky, 2004, 2005). Several findings support this working model. For example, estradiol treatment of gonadectomized male Wistar rats lowered the threshold to electrical kindling stimulation of the amygdala, an effect that could be mimicked by testosterone treatment but not DHT. Furthermore, in gonad-intact males that produced endogenous testosterone, aromatase inhibition blocked the progressive decrease in threshold typically observed over the kindling period (Edwards et al., 1999b). Similarly, testosterone treatment of male Sprague-Dawley rats was observed to increase susceptibility to seizures induced by either KA or pilocarpine injection (Mejias-Aponte et al., 2002), and gonadectomy of CF/1 mice increased susceptibility to seizures induced by combined PTZ and strychnine treatment, an effect that could be reversed by testosterone replacement (Pesce et al., 2000). In another study, testosterone treatment also increased seizure susceptibility in male rats and mice, and this seizure exacerbation could be blocked by treatment with the aromatase inhibitor, letrozole (Reddy, 2004c). Conversely, however, testosterone-treated wild-type mice exhibited an increased latency to systemic PTZ-induced seizures, but knockout mice lacking 5α-reductase did not show this effect of testosterone (Frye et al., 2001), indicating that the anticonvulsant effects of testosterone were likely mediated by 5α-reduction to DHT. DHT was also observed to be anticonvulsant against PTZ-induced seizures in male mice (Reddy, 2004c). It should be noted, however, that in studies examining the effects of gonadectomy and hormone replacement on excitatory synaptic spine density in the rat hippocampus, males exhibited a reduction in spine density following gonadectomy that was reversed by testosterone, but intriguingly, the effect of testosterone appeared to be mediated entirely by DHT (Leranth et al., 2003). This effect was in marked contrast to that of females, in which an upregulation of spine density by testosterone was almost completely blocked by letrozole, and DHT produced only a small effect (Leranth et al., 2004). Altogether, it appears that testosterone can exert robust effects on seizure susceptibility, and the end resulting effect depends critically on the balance of conversion to estradiol or DHT.

DHT can be further converted to the neurosteroid androstanediol (5α-androstan-3α,17β-diol, or 3α-Diol), which itself can be converted to androsterone (5α-androstan-3α-ol-17-one) (Kaminski et al., 2005; Reddy and Jian, 2010) (Fig. 3). Similar to progesterone-derived allopregnanolone, androstanediol acts as a positive allosteric modulator of GABA_ARs (Reddy and Jian, 2010) and is thus poised to exert anticonvulsant effects. Indeed, androstanediol treatment has been observed to be protective in various forms of seizure induction models, including hippocampal kindling, PTZ, picrotoxin, and β-carboline ester (Reddy, 2004a,c; Ryan and Frye, 2008; Frye et al., 2009; Reddy and Jian, 2010). There are conflicting reports regarding efficacy against KA-induced seizures (Frye and Reed, 1998; Reddy, 2004a), but these discrepancies may reflect species differences between rats and mice. Intriguingly, a recent study suggests that female mice are more sensitive to the anticonvulsant effects of androstanediol and that this sex effect reflects differences in the expression of δ subunit-containing GABA_ARs in dentate gyrus granule cells (Reddy et al., 2019). Similarly, androsterone treatment appears to produce antiseizure effects across multiple seizure models in male mice and rats, including 6-Hz corneal stimulation, PTZ, 4-aminopyridine, pilocarpine, and maximal electroshock (Kaminski et al., 2005), although actions of androsterone to lower seizure threshold in response to KA have been reported (Mroz et al., 2009). Overall, it appears that both androstanediol and androsterone exert potent anticonvulsant effects; thus, upregulating the downstream multistep conversion of testosterone to either androstanediol or androsterone, or direct treatment with these metabolic products, is a promising avenue of future ASD development. It should be noted, however, that the antiseizure effects of testosterone-derived neurosteroids outlined above may not entirely be the case for absence seizures, reflecting the mechanism of neurosteroid enhancement of inhibition producing stronger postinhibitory rebound burst firing in thalamocortical neurons (van Luijtelaar et al., 2014). In particular, it appears that testosterone itself (and/or DHT) may exert antiabsence effects, but androstanediol, particularly through enhancement of inhibition mediated by δ subunit-containing GABA_ARs, produces proabsence effects. In this regard, castrated male WAG/Rij rats, a genetic model of absence seizures, display more SWD than intact males, suggesting that on balance, the overall effects of testosterone are seizure-suppressive (van Luijtelaar et al., 1996).