GABA_A RECEPTORS AS BROADCASTERS OF SEXUALLY DIFFERENTIATING SIGNALS IN THE BRAIN

Abstract

Epileptic seizures are more common in males than in females. One of the areas that have recently been implicated in the higher susceptibility of males to seizures is the substantia nigra reticulata (SNR). Several studies support the existence of phenotypic differences between male and female infantile SNR neurons and particularly in several aspects of the GABAergic system, including its ability to control seizures. We have recently found that at postnatal day 14-17 (PN14-17) rats, which are equivalent to infants, activation of GABA_A receptors has different physiological effects in male and female SNR neurons. This is likely due to the differences in the expression of the neuronal-specific potassium-chloride cotransporter KCC2, which regulates the intracellular chloride concentration. In male PN14-17 SNR neurons, GABA_A receptor activation with muscimol causes depolarization and increments in intracellular calcium concentration and the expression of calcium regulated genes, such as KCC2. Blockade of L-type voltage sensitive calcium channels (L-VSCC) by nifedipine decreases KCC2 mRNA expression. In PN14-17 females, however, muscimol hyperpolarizes the SNR neurons, does not increase intracellular calcium and decreases KCC2 mRNA expression. In PN15 females, nifedipine has no effect on KCC2 mRNA expression in the SNR. This sexually dimorphic function of GABA_A receptors also creates divergent patterns of estradiol signaling. In male PN15 rats, estradiol decreases KCC2 mRNA expression in SNR neurons. Pretreatment with the GABA_A receptor antagonist bicuculline or with nifedipine, prevents the appearance of estradiol-mediated downregulation of KCC2 mRNA expression. In contrast, in PN15 females, estradiol does not influence KCC2 expression. These show that, in infantile rats, drugs or conditions that modulate the activity of GABA_A receptors or L-VSCCs have different effects on the differentiation of the SNR. As a result, they have the potency of causing long-term changes in the function of the SNR in the control of seizures, movement and the susceptibility to and course of epilepsy and movement disorders.

Introduction

The substantia nigra reticulata (SNR) is involved in the control of movement and seizures (1-17). Interestingly, the incidence of certain seizure and movement disorders is different in men and women. Epilepsy or unprovoked seizures are more common in males, although in most population studies this difference is small (18, 19). A potential confounding factor is the inclusion of patients with different ages and heterogeneous epileptic syndromes. When looking at specific syndromes, we encounter a male predominance in many pediatric syndromes, such as the severe and benign myoclonic epilepsies of infancy, or the autosomal dominant epilepsy with febrile seizures plus. Males also appear to be more vulnerable to acute symptomatic seizures (20, 21). In certain experimental models of epilepsy, sex differences in the susceptibility to certain types of seizures, seizure-induced damage, or response to antiepileptics have been reported (22-27).

A male predominance has also been reported in incidence studies on Tourette syndrome (28, 29) and Parkinson’s disease (30), which have been linked to an underlying dysfunction of the substantia nigra. Such observations have triggered a line of research to understand the pathophysiologic factors responsible for the higher susceptibility of men to these disorders. In this article, I will summarize the evidence supporting that the SNR is sexually dimorphic and will present our recent data that implicate GABA_A receptors as broadcasters of sexually differentiating signals, which may prepare the male and female SNR to assume different functional roles in controlling seizures and possibly movement.

Sexual dimorphism of rodent SNR

The differences between the male and female SNR probably start prenatally, with the peak of neurogenesis during embryonal life appearing earlier in female than in male rat SNR (31). Postnatally, sex differences in the expression of androgen and estrogen receptors emerge, and these may influence the responsiveness to the differentiating effects of sex hormones (32, 33). The dimorphic features of the GABA_A receptor system are particularly important, since the major synaptic input to the SNR is comprised of GABAergic synapses (34-36). Moreover, SNR neurons are primarily GABAergic (37) and contain GABA_A receptors (38-40). In vivo studies have demonstrated that the GABA_A-responsive SNR neurons of male and female rats have distinct physiological properties and functions. SNR neurons from anesthetized adult male rats develop tolerance to chronic benzodiazepine administration, as well as rebound increase in spontaneous firing upon administration of benzodiazepine antagonist, whereas those from ovariectomized females do not (41). Using intranigral infusions of the GABA_A receptor agonist in developing rats, Veliskova and Moshé have shown sex, age, and site-specific effects on the threshold to flurothyl seizures, which are altered by testosterone administration during the early postnatal life (42). But what are the mechanisms underlying these divergent functions? The explanations may be multifold, ranging from qualitative and quantitative differences in the expression of molecules that influence GABA_A receptor or other neurotransmitter signaling pathways, in the connectivity with other structures, in synaptic and morphological attributes of the GABAergic SNR neurons, as well as in humoral factors. Previous studies have documented sex differences in the GABAergic system of the rat SNR. For instance, the female SNR has higher GABA levels than the male (38, 43), whereas GABA turnover appears to be higher in males (44). The subunit composition of GABA_A receptors also differs, with an increased neuronal expression and relative abundance of the α1 subunit in female SNR neurons (38, 45). This may result in different pharmacological and physiological properties. Moreover, as the subsequent sections will demonstrate, GABA_A receptor activation elicits distinct physiologic responses in male and female SNR neurons.

Sex-specific KCC2 expression and functional maturation of GABA_A receptors in rat SNR

The initial report by Ben-Ari and colleagues (46) and many subsequent studies (47-51) have established the dual role of GABA_A receptors during development. Immature neurons have high intracellular chloride ([Cl⁻]_i) and as a result GABA_A receptor activation depolarizes them, activates voltage-sensitive calcium channels (VSCCs), and increases intracellular calcium ([Ca⁺⁺]_i). This results in activation of many calcium-regulated intracellular processes, including gene transcription and neuronal differentiation. Mature neurons, in contrast, have low [Cl⁻]_i and this allows GABA_A receptor agonists to cause neuronal hyperpolarization without activating VSCCs. Recently, a neuronal-specific isoform of the potassium chloride cotransporters, KCC2, was shown to be important facilitator of this functional switch, by controlling [Cl⁻]_i (52). Low expression of KCC2, as occurs in immature neurons, is not sufficient in lowering ([Cl⁻]_i) and as a result the increased [Cl⁻]_i facilitates depolarizing responses to GABA_A receptor activation. During development, KCC2 expression gradually increases in most brain structures, lowers [Cl⁻]_i and permits the appearance of hyperpolarizing responses to GABA_A receptor activation (52, 53).

To test whether similar functional differences in GABA_A receptor physiology occur between sexes, KCC2 expression in rat SNR was correlated with GABA_A receptor function. Using a KCC2 specific digoxigenin labeled in situ hybridization technique, sex, age and region specific differences in KCC2 mRNA expression were found in SNR neurons of PN15, PN30, and adult males and PN15 and PN30 female rats (54). Males always had lower KCC2 mRNA expression in their SNR compared with age-matched females. KCC2 mRNA expression increased between PN15 and PN30 in both sexes but plateaued thereafter in males. Anteroposterior differences were also present after PN30, with the anterior SNR neurons expressing more KCC2 mRNA compared with the posterior. Interestingly, comparison of KCC2 mRNA expression with the ability of intranigral muscimol infusions to elicit pro- or anticonvulsant effects in the flurothyl seizure model, as characterized by Veliskova and Moshé (42), revealed a very good correlation. The groups that responded to intranigral muscimol with proconvulsant effects had low KCC2 mRNA in their SNR, whereas the groups in which muscimol infusions had anticonvulsant effects expressed high KCC2 mRNA levels (54). These suggested that perhaps KCC2 regulates the sex, age and region-specific role of the SNR in seizure control by altering the electrophysiological properties of the SNR neurons. Indeed, in gramicidin-perforated patch clamp and fura-2AM imaging experiments, bath application of muscimol in acute SNR slices from male PN14-17 rats caused depolarizations and increments in [Ca⁺⁺]_i. In contrast, in females, muscimol-induced currents were hyperpolarizing and not associated with changes in [Ca⁺⁺]_i (54) (Figure 1). Since GABA_A receptor activation in adult male SNR neurons results in hyperpolarizing currents, these findings suggest that the functional maturation of the GABA_A receptors during development is delayed in males.

Figure 1. Sex differences in KCC2 and GABA_A receptor function in PN15 rat SNR.

Panels A and B: KCC2-specific digoxigenin labeled in situ hybridization of PN15 SNR sections. Male SNR neurons (panel A) express lower KCC2 mRNA than female (panel B) SNR neurons. Panel C: In PN15 male SNR, the low KCC2 expression neurons results in high intracellular chloride concentration. Upon GABA-mediated activation of GABA_A receptors, chloride efflux ensues, followed by depolarization, activation of voltage-sensitive calcium channels (VSCCs) and increase in intracellular calcium. Panel D: In PN15 female SNR neurons, the high KCC2 expression is sufficient to lower intracellular chloride. As a result, activation of GABA_A receptors leads to influx of chloride, hyperpolarization, without affecting the activity of VSCCs.

Dual effects of GABA_A receptors on calcium-regulated gene expression

Since GABA_A receptor activation regulates [Ca⁺⁺]_i in male but not in female infantile SNR, we hypothesized that this would result in sex-specific regulation of gene expression. Indeed, using KCC2 as a target gene, we have shown that modulators of GABA_A receptor or L-type VSCCs (L-VSCCs) have distinct regulatory effects on its expression in PN15 male and female SNR neurons in vivo (54). In males, muscimol increased whereas the L-VSCC blocker nifedipine decreased KCC2 mRNA expression in the SNR. These findings are in agreement with Ganguly et al (55) who showed that GABA can accelerate the developmental switch of GABA_A receptors. In PN15 female SNR neurons, however, muscimol decreased KCC2 mRNA but nifedipine had no effect (Figure 2).

Figure 2. Sexually dimorphic regulatory pathways affecting KCC2 mRNA expression in PN15 male and female rat SNR neurons.

In male PN15 rats, GABA_A receptor or L-type VSCC (L-VSCC) activation increases KCC2 mRNA expression, whereas estradiol downregulates KCC2 mRNA expression. The asterisk indicates that estradiol-mediated downregulation of KCC2 mRNA occurs only when GABA-mediated activation of L-VSCCs is present. In female PN15 rats however, GABA_A receptor activation downregulates KCC2 mRNA, whereas estradiol has no effect.

GABA_A receptors broadcast sexual differentiating signals in infantile rat SNR, in response to estradiol

To understand the regulatory mechanisms controlling KCC2 expression in rat SNR, we studied the effects of sex hormones (56). Interestingly, administration of 17β-estradiol downregulated KCC2 mRNA expression in the SNR of PN15 male rats within 4 hours but had no effect in females. These effects were already present within 4 hours, and persisted after repeated doses (PN13-15). The observation that regulation of KCC2 by GABA_A receptor induced increments in [Ca⁺⁺]_i and estradiol both occurred in males but not in females generated the hypothesis that perhaps there is an interaction or interdependence of these two signaling pathways. Indeed, pretreatment of male PN15 rats with blockers of GABA_A receptors, ie bicuculline, or with nifedipine, prevented the appearance of estradiol-mediated downregulation of KCC2 mRNA (56). Estradiol therefore requires the presence of L-VSCCs activated by GABA_A receptors in order to regulate KCC2 expression (Figure 2).

Sex differences in GABA_A receptor function in other brain regions

Divergent effects of GABA_A receptor activation have also been reported in the neonatal hypothalamus. Systemic injections of muscimol in neonatal rats (PN0) increase the expression of the phosphorylated form of the cAMP responsive element binding protein (phospho-CREB) in the ventromedial hypothalamus, the medial preoptic area and the CA1 area of the hippocampus but not in the arcuate nucleus (57). The muscimol-induced increase in phospho-CREB in these areas was inhibited by pretreatment with the L-type voltage sensitive calcium channel blocker nimodipine (58). These observations could suggest that a similar sexual dimorphism in the function of native GABA_A receptors and expression of cation chloride cotransporters may exist in some of these structures. To date, studies in neonatal pups of male or unspecified sex have provided ample evidence that GABA_A receptor activation elicits depolarizing responses in the neonatal life in both these structures and hyperpolarizing effects later on during development (59, 60). Persistence however of depolarizing effects of GABA in certain populations of adult hypothalamic cells have been reported, which correlate with absence or low expression of KCC2 in these cells (61) (62). Correlation of GABA_A receptor function and electrophysiology with chloride cotransporter expression in specific subpopulations of cells of the same model system would be needed to derive definite conclusions as to the existence of sex differences in structures other than the SNR.

Conclusions

These data provide the first evidence that the expression of a cation-chloride cotransporter, KCC2, changes not only as a function of age but also of sex. Although direct modulation of KCC2 expression in the SNR may be necessary to firmly establish a cause and effect relationship between sex differences in KCC2 expression and GABA_A receptor function, this is strongly supported by the existing literature (52, 53). The ensuing differences in GABA_A receptor physiology appear to influence the sex, age and region specific role of the SNR in seizure control. It would be interesting to speculate whether KCC2 or other related molecules that regulate chloride homeostasis may be implicated in pediatric epilepsy syndromes, especially those that exhibit male preponderance. To this end, it would also be worth examining whether similar sex differences in cation chloride cotransporters and GABA_A receptor function exist in other cortical and subcortical structures involved in seizure generation and propagation.

Male SNR neurons retain the “immature type” phenotype of GABA_A receptors for longer developmental periods compared to females. As a result, activation of GABA_A receptors in male SNR neurons may have long term trophic, proliferating, and differentiating effects, promoting thus the sexual differentiation of the SNR and its role in seizure or movement control (63, 64). Furthermore, the use of drugs acting on GABA_A receptors or L-VSCCs, as many antiepileptic agents do, or conditions that modulate these receptor systems, such as seizures, may have different long term differentiating effects on the male and female SNR, during vulnerable developmental periods. The dependence of estradiol-mediated regulation of KCC2 expression upon the presence of GABA-mediated activation of L-VSCCs is especially relevant since sex hormones, and estrogens in particular, have been implicated in the differentiation and masculinization of the brain (33). First, our findings suggest that one of the factors defining the critical period during which certain structures, such as the SNR, and certain calcium-regulated genes, such as KCC2, are sensitive to the differentiating and trophic effects of estrogens may indeed be the presence of GABA-mediated depolarization and calcium rises. Second, during developmental periods characterized by sexually dimorphic functions of GABA_A receptors, such as the PN14-17 rat SNR, GABA_A receptors broadcast different signals in male and female neurons responding to estradiol, further promoting the sexual differentiation of the structure.

Finally, the dual regulation of KCC2 mRNA by GABA_A receptor agonists, under conditions that they may trigger depolarizing or hyperpolarizing responses, may indeed act as a feedback mechanism to prevent excess excitation or inhibition when prolonged or excessive stimulation of GABA_A receptors occurs. Further documentation at the protein level or electrophysiology is however needed.