Sex differences in behavioral pathology induced by subconvulsive stimulation during early postnatal life are overcome by epileptic activity in the pre-juvenile weanling period.

Chronic subconvulsive activity in early life leads to sex-related autistic-like deficits in handling, object recognition, and social performance in pre-pubertal rats. Since autism and epilepsy are common neurodevelopmental disorders with high coincidence, we tested whether early-life chronic subconvulsive activity compared to convulsive activity alters handling, spatial memory, lateralization, coping strategy and the seizure threshold in a sex-dependent manner. A hypothesis is that convulsive seizures may alter sex differences induced by subconvulsive (SC) activity. Serial subconvulsive doses of kainic acid (KA) were administered postnatally (0.25-1 mg/kg) for 15 days to induce the chronic subconvulsive phenotype (SC group). Age-matched controls and a subset of SC pups were exposed to a convulsive dose of KA (KA and SC + KA groups; 7.5 mg/kg) or flurothyl vapors. In our open handling test, controls and the ASD groups escaped to a similar degree whereas after convulsive seizures, the pups exhibited freezing behavior; no escapes occurred. In the spontaneous alternating T-Maze control males and females entered the left arm with higher frequency. The SC males but not SC females entered left and right arms to a similar degree; alternation rates were reduced to chance revealing a sex difference. However, in KA and SC + KA groups, there was a sharp loss of spontaneous alternation rates. The rapid repetitive entries shifted to the right in both sexes possibly be due to hippocampal injury and changes in network activity induced by status epilepticus. In the forced swim test (FST), control and CS females were more active than corresponding males. After convulsions, immobility was reduced and vertical mobility was increased in SC and SC + KA males suggesting an elevated coping strategy compared to females. Onset and severity of KA induced status epilepticus was delayed in SC males and females possibly due to desensitization of KA receptors. Following flurothyl exposure, control males had faster onset of twitches and clonic seizures than control females which disappeared after the sub-convulsive pre-treatment. Data suggest that behavioral manifestations are more readily detectable between males and females when low levels of hyperexcitation are present chronically in early postnatal development but diminished after tonic-clonic convulsions persist. Therefore, therapeutic interventions may benefit patients if initiated upon the initial onset of sex-related autistic pathologies, particularly in males, which may reduce subsequent vulnerability to seizures.

The role of estrogens in seizures and epilepsy: the bad guys or the good guys?

Estrogens influence neuronal activity and are important for normal brain functions. Effects of estrogens on seizures are contradictory. It is commonly accepted that estrogens may increase neuronal excitability and thus mediate proconvulsant effects. However, clinical and animal data show that estrogen may also have no effect or anticonvulsant effects. The action of estrogens on seizures depends on various factors, such as treatment duration and latency prior to the seizure testing, estrogen dose, hormonal status (naïve vs gonadectomized animals), estrogenic substance, the region/neurotransmitter system involved, the seizure type/model used, and sex. Besides the effects on seizure susceptibility, estrogens may also play an important role in seizure-induced damage. Pretreatment with beta-estradiol in ovariectomized female rats has neuroprotective effects on status epilepticus-induced hippocampal damage and prevents the loss of inhibition in the dentate gyrus during the early post-status epilepticus period determined by the in vitro paired pulse paradigm. Several signaling pathways may be involved in the neuroprotective effects of beta-estradiol on status epilepticus-induced hippocampal damage but at least one of these pathways involves interactions with neuropeptide Y.

Oestradiol Regulates Neuropeptide Y Release and Gene Coupling with the GABAergic and Glutamatergic Synapses in the Adult Female Rat Dentate Gyrus.

Neuropeptide Y (NPY) is an endogenous modulator of neuronal activity affecting both GABAergic and glutamatergic transmission. Previously, we found that oestradiol modifies the number of NPY immunoreactive neurones in the hippocampal dentate gyrus. In the present study, we investigated which oestrogen receptor type is responsible for these changes in the number of NPY-positive neurones. Furthermore, we determined the effects of oestrogen receptor activation on NPY release. Finally, we examined the contribution of oestrogen toward the remodelling of the GABAergic and glutamatergic gene networks in terms of coupling with Npy gene expression in ovariectomised rats. We found that activation of either oestrogen receptor type (ERα or ERß) increases the number of NPY-immunopositive neurones and enhances NPY release in the dentate gyrus. We also found that, compared to oestrogen-lacking ovariectomised rats, oestrogen replacement increases the probability of synergistic/antagonistic coupling between the Npy and GABAergic synapse genes, whereas the glutamatergic synapse genes are less likely to be coupled with Npy under similar conditions. The data together suggest that oestrogens play a critical role in the regulation of NPY system activity and are also involved in the coupling/uncoupling of the Npy gene with the GABAergic and glutamatergic synapses in the female rat dentate gyrus.

Sex differences in androgen and estrogen receptor expression in rat substantia nigra during development: an immunohistochemical study.

Gonadal hormones are important regulators of sexual differentiation of the CNS. Exposure to testosterone and estrogen during development causes permanent organizational differences between males and females. We previously described functional sex-related differences of the GABA(A)ergic circuits of the rat substantia nigra pars reticulata (SNR) involved in the control of flurothyl seizures. This sexual differentiation of the SNR is regulated by postnatal testosterone. To assess whether the organizing effects of testosterone in the SNR are mediated via the androgen receptor (AR) and/or estrogen receptors (ER), we used immunohistochemistry to study the ontogeny of AR, ERalpha and ERbeta expression in SNR and substantia nigra pars compacta (SNC) of male and female rats. Rats on the day of birth [postnatal day (PN) 0] and at PN1, PN5, PN15 and PN30 were used. AR- and ERbeta-immunopositive cells were present in SNR and SNC in both sexes and at all ages. ERalpha was not detected in male and female SNC at PN0-PN1. In both substantia nigra (SN) regions, there were developmentally regulated sex differences in AR, ERalpha and ERbeta immunoreactivity. In the SN, each receptor showed specific intracellular localization: AR was present in the nucleus, ERalpha and ERbeta were present both in nuclear and extranuclear compartments. ERalpha was detected also in processes. At PN0-PN1, quantitative analysis revealed sex and regional differences in the distribution of SN cells expressing AR and ERalpha, while ERbeta were equally present in both sexes. The presence of gonadal steroid receptors in the SN suggests that the biological effects of gonadal hormones in the CNS extend beyond reproduction-related functions and may affect and modify motor behaviors (including seizures) in a sex-specific manner. Based on the ontogeny of SNR ERbeta, we hypothesize that postnatal injections of testosterone may regulate the nigral GABA(A) system through the aromatization pathway and activation of ERbeta.

GluR2 hippocampal knockdown reveals developmental regulation of epileptogenicity and neurodegeneration.

In adult rats, kainic acid-induced status epilepticus reduces GluR2 subunit expression prior to neurodegeneration of hippocampal CA3 neurons. Increased formation of Ca2+ permeable AMPA receptors may contribute to the delayed neurodegenerative process. In rat pups, highly prone to seizures but resistant to seizure-induced hippocampal damage, GluR2 mRNA and protein expression remain constant in CA3 neurons possibly contributing to their survival. To investigate whether reduced GluR2 expression in hippocampus may lead to enhanced hippocampal vulnerability in an age-dependent manner and whether changes correspond to altered electroencephalography (EEG) patterns, unilateral microinfusion of GluR2 antisense oligodeoxynucleotides (AS-ODNs) into hippocampus was performed at three ages (postnatal (P8), P13, and adult). At P13, GluR2 knockdown resulted in spontaneous seizure-like behavioral manifestations and neurodegeneration of CA3 neurons lateral and distal from the cannula infusion site. EEG recordings revealed high rhythmic activity associated with seizure-like behavior. In P8 pups and adult rats, there were no behavioral manifestations; distant hippocampal damage of the CA3 was not observed. Results indicate that unilateral knockdown of hippocampal GluR2 subunit expression induces age-dependent seizure-like behavioral manifestations, altered EEG recording patterns, and reduces the survival of CA3 neurons in the hippocampus of young rats during a specific postnatal period (3rd week), when GluR2 expression peaks in development and glutamatergic inputs are maturing.

Subthalamic nucleus: a new anticonvulsant site in the brain.

Several brain sites are important for seizure control, including the area tempestas, superior colliculus, thalamus and substantia nigra. These sites are especially sensitive to changes in GABAergic transmission achieved by localized infusions of GABAergic agents. We investigated the effects of the GABAA receptor agonist muscimol (100 ng in a volume of 0.25 microliter) microinfused into the subthalamic nucleus (STN) in the flurothyl seizure model in male adult rats. Both bilateral symmetrical and unilateral STN infusions of muscimol had a significant anticonvulsant effect against flurothyl-induced clonic seizures. Our data suggest that the STN is a part of the network responsible for the control of clonic seizures.

Site-specific effects of local pH changes in the substantia nigra pars reticulata on flurothyl-induced seizures.

Local cerebral changes of acid-base balance may interfere with neuronal communication. Acidosis enhances and alkalosis suppresses GABAA receptor neurotransmission while there are opposite effects on NMDA receptor transmission. In this study, we determined site-specific effects of acidified solutions of Na-HEPES-artificial cerebrospinal fluid infused into the anterior or posterior area of the substantia nigra pars reticulata (SNR) in rats. Two levels of pH were compared: 6.7 and 7.4. Rats were challenged with flurothyl and the threshold for clonic and tonic-clonic seizures was determined. In the anterior SNR, there were no differences between the effects of the solution with pH 6.7 and 7.4 on flurothyl seizures. In contrast in the posterior SNR, microinfusions with pH 6.7 had proconvulsant effects. The results suggest that local pH changes may have site-specific effects on seizure susceptibility in vivo.

Prenatal morphine exposure alters ovarian steroid hormonal regulation of seizure susceptibility.

The present study examined the ovarian hormonal regulation of seizure susceptibility in prenatally morphine- and saline-exposed adult female rats in the flurothyl seizure model in vivo, and in low-magnesium-induced epileptiform activity in brain slices, in vitro. All females were ovariohysterectomized (OVX); some received either estrogen (E) or progesterone (P) replacement, while others were injected with E + P sequentially. In prenatally saline-treated control females, there was an increase in the flurothyl-induced clonic seizure threshold (anticonvulsant effect) in the presence of both hormones (E + P) compared to OVX controls. In morphine-exposed females, there was an increase in the flurothyl-induced clonic seizure threshold after an E injection alone while there was a reduced tonic--clonic seizure threshold in the presence of both hormones (E + P) compared to the hormone treatment-matched group of saline-exposed females. In control females, in low magnesium medium in vitro, the development of two types of epileptiform activity (seizure-like events and status of short discharges) was not affected by the different hormonal conditions. However, prenatal morphine exposure suppressed the development of both types of epileptiform activity in the E-injected females compared to the E-injected, control females. The present data demonstrate that the anticonvulsant effects of P on seizure susceptibility requires the presence of E. Furthermore, prenatal morphine exposure alters ovarian steroid hormone-regulated seizure susceptibility.

Maturation and segregation of brain networks that modify seizures.

The mature brain is less susceptible to seizures than the immature brain. We demonstrate that in the mature substantia nigra (SN) there are two topographically discrete GABAA-sensitive regions which differ in the amount of mRNA expression of the GABAA receptor alpha 1 subunit. These two regions mediate separate anticonvulsant and proconvulsant effects and use divergent projection networks. By contrast, in the immature SN there is no special topography of mRNA expression of the alpha 1 subunit and only the proconvulsant network is present. The decreased seizure susceptibility of the mature brain may be related to postnatal segregation of GABAA-sensitive networks.

Region-specific modulation of limbic seizure susceptibility by ovarian steroids.

Gonadal steroid hormones can markedly affect seizure susceptibility. Ovariohysterectomized female rats given ovarian steroid hormone supplements were used to evaluate the effects of ovarian steroids on epileptiform activity in hippocampal slices in vitro and on flurothyl-induced seizures in vivo. Seizure susceptibility was compared in the entorhinal cortex (EC) and CA1 regions of the hippocampus perfused with Mg(2+)-free medium, which leads to epileptiform discharges caused by a relief of voltage-dependent NMDA receptor block. After in vivo treatment with 500 microg of progesterone for 2 h prior to slice preparation, the latency to onset of low Mg(2+)-induced epileptiform activity of slices was significantly prolonged compared to slices from controls. In contrast, progesterone replacement accelerated the development of epileptiform activity in the CA1 region. Neither estrogen alone (2 x 2 microg of estradiol benzoate, 48 and 24 h prior to the experiment), nor a combined treatment with estrogen plus progesterone, significantly affected seizure susceptibility in either CA1 or the EC. There were no consistent effects of estrogen or progesterone, alone or in combination, on flurothyl-induced seizures in vivo. The data suggest that in vitro, progesterone alters seizure susceptibility in a site- and seizure model-specific fashion. The differential effects of progesterone may be due to differential expression of progesterone receptor isoforms or metabolites in specific brain areas suggesting that selective modulation of NMDA receptor-dependent epileptiform activity may play a role in hormonal effects on epileptogenesis.

Developmental regulation of regional functionality of substantial nigra GABAA receptors involved in seizures.

GABAergic (gamma-aminobutyric acid) transmission in the substantia nigra pars reticulata is critical for seizure control. We tested the hypothesis that there is a differential regional distribution and functionality of nigral GABAA receptor sites that is developmentally regulated. In adult rats, we determined the effects on flurothyl seizures of (Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid (ZAPA, a presumed agonist of the low-affinity GABAA receptor site), bicuculline (an antagonist of the low-affinity GABAA receptor site) and gamma-vinyl-GABA (a GABA-transaminase inhibitor), infused bilaterally in anterior or posterior substantia nigra pars reticulata. ZAPA infusions (8 micrograms) were anticonvulsant in anterior substantia nigra but proconvulsant in posterior substantia nigra. Bicuculline infusions (100 ng) were proconvulsant in anterior substantia nigra but ineffective in posterior substantia nigra. An anticonvulsant dose of gamma-vinyl-GABA, when infused in anterior substantia nigra, was proconvulsant when infused in posterior substantia nigra. In 15 day old rats, the effects of ZAPA, were biphasic: 2 micrograms was anticonvulsant while 8 micrograms was proconvulsant. There was no regional specificity. The data suggest that with maturation there is functional segregation of specific GABAA receptor subtypes involved in substantia nigra-mediated seizure control.

Chronic neonatal phencyclidine treatment produces age-related changes in pentylenetetrazol-induced seizures.

Although excitatory amino acids are known to play a critical role in the plasticity of developing brain, the behavioral effects of blocking the N-methyl-D-aspartate (NMDA) receptor-gated ion channel during development are not clear. Here we report the effects of chronic postnatal administration of 1-phenylcyclohexylpiperidine (phencyclidine or PCP), a NMDA channel blocker, on seizure susceptibility. To study the short-term effects of chronic PCP administration on pentylenetetrazol (PTZ)-induced seizures, rats were treated with PCP (5 mg/kg, i.p.) for 11 days from postnatal days 5-15, 24-34 or 44-54 and tested in the PTZ-induced seizure paradigm on postnatal days 21, 40 and 60, respectively. Administration of PCP in 5-15-day-old rats resulted in increased seizure susceptibility at day 21, while administration of PCP in postweanling rats (days 24-34) markedly attenuated their susceptibility to seizures at day 40. PCP injection had little effect on the seizure susceptibility of older rats. To study the long-term effects of postnatal PCP treatment, rats were injected with PCP (5 mg/kg from postnatal day 5-15, i.p.) and were tested for PTZ-induced seizures on postnatal days 40 and 60; each rat was tested only once. When tested for PTZ-induced seizure on day 40, PCP-treated rats did not differ from saline-treated controls. When tested on day 60, PCP-treated rats had a lower incidence of seizures and in the rats that did have seizures their latencies were significantly prolonged compared to controls. Together, our data suggest that chronic PCP administration alters PTZ-induced seizure susceptibility in an age-dependent manner and chronic PCP administration in postnatal rats produces long-term changes that persist into adulthood.

Prenatal morphine exposure differentially alters seizure susceptibility in developing female rats.

We examined the effect of prenatal morphine exposure (5-10 mg/kg on days 11-18 of gestation) on seizure susceptibility in female rats during development. The effect of morphine exposure on flurothyl-induced seizures was age-dependent. At postnatal day (PN) 15, morphine exposure decreased both clonic and tonic-clonic seizure thresholds compare to saline controls. At PN 25, morphine exposure did not alter the clonic seizure threshold but increased the threshold to tonic-clonic seizure. At PN 38, morphine exposure did not influence either threshold. The data suggest that the effects of prenatal exposure to opioids on seizures are age-related and transient.

Prenatal morphine exposure alters susceptibility to bicuculline seizures in a sex- and age-specific manner.

Bicuculline was used to investigate seizure susceptibility in pre- and peripubertal male and female rats exposed prenatally to morphine. Morphine-exposed males showed increased seizure susceptibility at prepubertal and decreased susceptibility at peripubertal ages. There was no difference in seizure susceptibility in morphine-exposed females at either age. Therefore, the present data suggest that males are more vulnerable than females to morphine-induced insults during prenatal brain development.

Further evidence of involvement of substantia nigra GABAB receptors in seizure suppression in developing rats.

To determine whether the substantia nigra GABAergic anticonvulsant effects depend on GABAB receptor activation, we tested the effects of intranigral CGP 35,348 (a GABAB receptor antagonist) alone or in combination with gamma-vinyl-GABA (GVG; a GABA-transaminase inhibitor) on flurothyl seizures in rat pups and adult rats. In pups, nigral infusions of CGP 35,348 decreased the thresholds for clonic and tonic seizures and attenuated the anticonvulsant effects of GVG. In adults, nigral infusions of CGP 35,348 did not alter seizure thresholds. The data suggest that, in rat pups, nigral GABAB receptors regulate, in part, flurothyl-induced seizures.

The effects of N-methyl-D-aspartate antagonist 2-amino-7-phosphonoheptanoic acid microinfusions into the adult male rat substantia nigra pars reticulata are site-specific.

We examined the effects of regional infusions of 2-amino-7-phosphonoheptanoic acid (AP7) into the substantia nigra pars reticulata (SNR) in adult male rats on flurothyl-induced clonic seizures. AP7 infusions in the SNR had dose- and region-specific effects. In the SNR(anterior), both doses of AP7 (0.1 and 5 nmol) produced an anticonvulsant effect. In the SNR(posterior), the lower dose (0.1 nmol) did not influence the seizure threshold whereas the higher dose (5 nmol) of AP7 had a proconvulsant effect. The data indicate that there are two distinct regions within the SNR which differentially respond to pharmacological manipulations of the glutamatergic system. The region-specific effects in the SNR may provide an explanation for the previous conflicting reports regarding infusions of N-methyl-D-aspartate antagonists into this structure.

Testosterone regulates androgen and estrogen receptor immunoreactivity in rat substantia nigra pars reticulata.

At postnatal day (PN)1, there are sex differences in gonadal receptor expression in the rat substantia nigra pars reticulata (SNR). Male pups have lower levels of androgen receptor (AR) and estrogen receptor (ER)beta immunoreactivity (IR) compared to female pups, while ERalpha IR is equally expressed in the two sexes. To test whether these differences are due to sex differences in testosterone exposure, we injected female pups with testosterone propionate (TP) on the day of birth and analyzed the levels of AR and ER IR at PN1. TP-treated females have lower levels of AR and ERbeta IR than control, while there are no differences in the levels of ERalpha IR. TP treatment did not affect the number of AR and ER expressing cells. The regulation of SNR AR and ERbeta IR by testosterone may be important for the development of sex-specific functional systems involved in motor control.

Bicuculline-induced neocortical epileptiform foci and the effects of 6-hydroxydopamine in developing rats.

Catecholamines (dopamine and norepinephrine) are considered to be predominantly inhibitory neurotransmitters in the brain and their depletion produced by 6-hydroxydopamine may result in proconvulsant effects. In our experiments on rats aged 5, 7, 9, 12, 15, 18, 25 and 90 days under urethane anesthesia we demonstrated the development of neocortical epileptic focus evoked by topical application of bicuculline methiodide. In experimental groups aged 7, 12, 18, 25 and 90 days a chronic depletion of catecholamines was induced using pretreatment with 6-hydroxydopamine early postnatally. An epileptogenic focus was induced in all age groups; duration of a single discharge decreased with age in both control and experimental animals. The spread of activity from the primary focus to contralateral frontal cortex via callosal connections was as rapid as in controls. However, the transfer of discharge to occipital regions was delayed and the number of discharges decreased in experimental rats. Our study demonstrated a substantial role of catecholamines for synchronization of focal discharges in neocortex and a promoting role of catecholamines in association pathways within neocortex.

Regional and age specific effects of zolpidem microinfusions in the substantia nigra on seizures.

GABAergic (gamma-aminobutyric acid) transmission in the substantia nigra pars reticulata (SNR) is critical for seizure control. The SNR effects on seizures are site-specific within the SNR and developmentally regulated. These age- and site-specific effects may be due to differential regional distribution and functionality of SNR GABA(A) receptor sites. We investigated the role of GABA/benzodiazepine (BZD) receptors in the SNR in the control of seizures as a function of age. In adult rats, we determined the effects of bilateral zolpidem (an agonist of the BZD1 receptor site) microinfusions in the anterior or in the posterior SNR (SNRanterior or SNRposterior, respectively) on flurothyl-induced clonic and tonic-clonic seizures. In SNRanterior, zolpidem microinfusions were anticonvulsant but ineffective in SNRposterior against clonic seizures. Microinfusions of zolpidem in SNRposterior or above SNR, did not alter the threshold to clonic seizures. SNR microinfusions of zolpidem did not alter the threshold to tonic-clonic flurothyl-induced seizures. In 15 day old (PN 15) rats, the SNR microinfusions of zolpidem had anticonvulsant effects on clonic and tonic-clonic seizures. There was no regional specificity. Microinfusions of zolpidem above the SNR, did not alter the threshold to clonic or tonic-clonic seizures. Our data demonstrate that the BZD1 binding sites are involved in the SNR control of flurothyl seizures in adult and PN 15 male rats.

Age-dependent vulnerability to seizures.

Seizure disorders frequently occur early in life. Seizures are classified as reactive, symptomatic, or idiopathic depending on whether their cause can be identified. Reactive seizures are the result of acute environmental perturbations. Early in life, many stressors can produce seizures and the ultimate outcome may depend on the particular precipitating factor and its intensity. Febrile convulsions are the most common reactive seizures, although they must be differentiated from symptomatic seizures precipitated by fever. Symptomatic seizures are often associated with varying degrees of central nervous system (CNS) insults, including congenital malformations and metabolic storage diseases of the gray matter. These seizures may have age-specific characteristics and may at times be difficult to treat with conventional antiepileptic treatments. To develop a better understanding of the pathophysiology of seizures early in life, we have extensively used animal models of epilepsy. In this chapter, we report our findings with a rat model of developmental cortical dysplasias produced by intrauterine injections of methylazoxymethanol acetate. These rats are more susceptible to kainic acid, flurothyl, and hyperthermic seizures than normal rats. Rats with severe cortical dysplasia are most susceptible to seizures. We have also studied the mechanisms involved in the control of seizures during development because status epilepticus is more prevalent in infants than in adults. Our data suggest that the substantia nigra may play a crucial role in status epilepticus as a function of age. In the adult substantia nigra two regions mediate opposing effects on seizures following infusions of gamma-aminobutyric acid type A (GABAA) agents. One region is located in the anterior substantia nigra, and muscimol infusions in this region mediate anticonvulsant effects. The second region is in the posterior substantia nigra, and here muscimol infusions produce proconvulsant effects. In situ hybridization data demonstrate that, at the cellular level, neurons in the two substantia nigra regions differ in the amount of hybridization grains for GABAA receptor alpha 1 and gamma 2L subunit mRNAs. In developing male rats, only the "proconvulsant" region is present up to the age of 21 days. The transition from the immature to mature substantia nigra mediated seizure control occurs between age 25 and 30 days. The identification of age-dependent functional networks involved in the containment of seizures may lead to possible new pharmacologic strategies to control seizures, thus aiding the development of age-appropriate treatments of seizure disorders.